CHARACTERIZATION OF THE REACTIONS
INVOLVED IN THE CONVERSION; OF
ORNITHINE TO 2-AMINO-4-
KETOPENTANOIC ACID IN
CLOSTRIDIUM STICKIAND II

Thesis for the Degree of Ph. D.
RICHIGAN STATE UNIVERSITY
RALPH SOMACK
1972

     
  

 

fi‘fi‘ 4

LIBRAR '9:
MichiganS ; ‘

University

  

This is to certify that the

thesis entitled

"CHARACTERIZATION OF THE REACTIONS INVOLVED IN
THE CONVERSION OF ORNITHINE TO
2-AMINO-4-KETOPENTANOIC ACID IN

CLOSTRIDIUM STICKLANDII"

presented by

Ralph L. Somack

has been accepted towards fulfillment
of the requirements for

Ph. D. degreein Microbiology and
Public Health

 

Date W 2

0-763!

       

3 I swim; I"
NOAH 8 SONS'
BOOK BINOERY INO'

L BRARY BINDERS ~
gaming. MlcmsA! '

 
       

Illk

 

ABSTRACT

CHARACTERIZATION OF THE REACTIONS INVOLVED IN THE
CONVERSION OF ORNITHINE IT) 2-AMINO-4—KETOPENTANOIC
ACID IN CLOSTRIDIUM STICKLANDII

 

BY

Ralph Somack

The first intermediate in the oxidation of ornithine by
Clostridium sticklandii, 2,4-diaminopentanoic acid, has been purified
from cell extracts in gram quantities. The dibenzoyl derivative and
the monopicrate and dihydrochloride salts were crystallized and
characterized. These compounds were employed to verify the structure
of the new amino acid by infrared, mass, and nuclear magnetic
resonance spectrometry.

The NAD+-NADP+-dependent 2,4-diaminopentanoic acid C

4

dehydrogenase from Clostridium sticklandii has been purified to

 

homogeneity by the criteria of disc gel electrophoresis and
ultracentrifugation. The weight average molecular weight of the
native enzyme as determined by high speed sedimentation equilibrium
is 72,000. Sedimentation velocity indicated an 520,w of 4.478.

Sodium dodecyl sulfate disc gel electrophoresis and high speed

 

sedimentation in 6 M guanidine-HCl established that the enzyme

is composed of two subunits of identical size. The enzyme is

Ralph Somack

sensitive to thiol inhibitors and titration with 5,5'-dithiobis-
2-nitrobenzoic acid and p-chloromercuribenzoate demonstrated the
presence of six sylfhydryl groups per mole. Amino acid analysis
indicated that the enzyme contains six half-cystine residues per
mole.

The coenzyme B -dependent ornithine mutase from Clostridium

12

sticklandii catalyzing the conversion of ornithine to 2,4-diamino-

 

 

pentanoic acid (2,4-DAP) has been purified to homogeneity. A radio-
chemical assay employing 14C-labeled ornithin e and a rapid, coupled
spectrophotometric assay employing 2,4-diaminopentanoic acid C4
dehydrogenase are described. Analysis by gel electrophoresis,

sucrose gradient centrifugation and SDS gel electrophoresis indicated
that the mutase has a molecular weight of about 180,000 and consists
of 2 subunits of identical size. The enzyme is specific for
D~a-ornithine and is inhibited by L-u-ornithine, DL-u-lysine, and
B-lysine. Kinetic and inhibitor studies showed that ornithine

mutase and C4 dehydrogenase are directly linked and that pyridoxal
phosphate is a cofactor for ornithine mutase. The absorption spectrum
of the mutase measured directly in analytical gels indicated that a
substantial amount of native bound cobamide had been converted to
inactive hydroxocobalamins. After incubation with B coenzyme and

12

subsequent dialysis, the spectrum was more typical of bound 312
coenzyme. The ornithine mutase reaction is reversible and proceeds
to approximately an equal extent in both directions. However, the

product, 2,4-DAP, appeared to inhibit the reverse reaction at

Ralph Somack

concentrations greater than 0.7 mM when present alone. The enzyme
contains labile sulfhydryl groups and is inhibited by oxygen.

Experiments with H O-t indicated that the reaction proceeds by a

2

mechanism which excludes exchange of hydrogen with the solvent.

CHARACTERIZATION OF THE REACTIONS INVOLVED IN THE

CONVERSION OF ORNITHINE It) 2-AMINO-4-KETOPENTANOIC

ACID IN CLOSTRIDIUM STICKLANDII

 

By

,a
.‘9

RalphfSomack

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Microbiology and Public Health

1972

TABLE OF CONTENTS

LIST OF TABLES

 

LIST OF FIGURES . . . .
INTRODUCTION
LITERATURE REVIEW . . . . . . . . . . . . . . . .
Part
I. The Amino Acid Fermenting Clostridia
II. The Fermentation of Ornithine and Lysine
Introduction

Fermentation of ornithine .
Fermentation of lysine . . . . . . .

III. Other Coenzyme B -dependent Mutases

12

Introduction

Carbon-nitrogen bond cleaving mutases .

Carbon-carbon bond cleaving mutases .

Carbon-oxygen bond cleaving mutases .

IV. Vitamin BIZ-dependent Reactions .
V. Mechanisms of BIZ-dependent Reactions .
REFERENCES

ii

Page

iv

10
10
11
15
16
17
19

22

Page
ARTICLE 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Preparation and Characterization of 2,4-Diaminova1eric
Acid: An Intermediate in the Anaerobic Oxidation of
Ornithine. R. L. Somack, D. H. Bing, and R. N. Costilow.
Analytical Biochemistry, Vol. 41, Number 1, May 1971.
ARTICLE 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

2,4-Diamin0pentanoic Acid C Dehydrogenase: Purification

4
and Properties of the Protein. R. L. Somack and
R. N. Costilow. J. Biol. Chem. (In Press), 1972.

ARTICLE 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Purification and PrOperties of a Pyridoxal Phosphate
and BIZ-Coenzyme—dependent D-a-Ornithine-4,5-

Aminomutase. R. L. Somack and R. N. Costilow. Manu-

script to be submitted to Biochemistry.

 

iii

 

TABLE

LIST OF TABLES

LITERATURE REVIEW

812 Coenzyme-dependent Reactions
Properties of Coenzyme BIZ-dependent Mutases

ARTICLE 1
Chromatographic and Electrophoretic Properties of

2,4-Diaminova1eric acid-ZHCI

ARTICLE 2

Purification of C4 Dehydrogenase
(Supplementary Results)

Amino Acid Analysis of C Dehydrogenase .

4
Effect of Sulfhydryl Inhibitors .
ARTICLE 3
Purification of Ornithine Mutase
Effect of Compounds Structurally Related to Ornithine
on Mutase Activity
Effect of Sulfhydryl Inhibitors on Mutase Activity

Effect of Dithiothreitol and Oxygen on Mutase

Activity

iv

Page

12

14

30

51

56

57

91

92

93

94

LIST OF FIGURES

FIGURE Page

LITERATURE REVIEW

 

 

1. Ornithine and lysine fermentation in Clostridium
sticklandii . . . . . . . . . . . . . . . . . . . . . 6
ARTICLE 1
1. Infrared absorption spectrum of 2,4-diaminova1eric
acid dihydrochloride (KBr pellet) . . . . . . . . . . 31
2. 100 MHz nuclear magnetic resonance spectrum of

2,4-diaminova1eric acid dihydrochloride

(020 solution, TMS as external reference) . . . . . . 32
ARTICLE 2
l. Preparative disc gel electrophoretic elution profile
of C4 dehydrogenase . . . . . . . . . . . . . . . . . 53

(Supplementary Results)

1. High speed equilibrium centrifugation of C4
dehydrogenase . . . . . . . . . . . . . . . . . . . . 59
ARTICLE 3
1. Disc gel electrophoresis of pure ornithine mutase . . . . 96
2. Sucrose density gradient centrifugation of ornithine
mutase . . . . . . . . . . . . . . . . . . . . . . . 98
3. SDS gel electrophoresis of pure ornithine mutase . . . . 100

 

 

 

10.

FIGURE

10.

Temperature Optimum of ornithine mutase .

pH optimum or ornithine mutase

Lineweaver-Burk plot for D-ornithine with pure
ornithine mutase

Lineweaver-Burk plot for pyridoxal phosphate with
pure ornithine mutase .

Conversion of ornithine to 2-amino-4-ketopentanoic
acid by mutase and C4 dehydrogenase .

Evidence for mutase-bound cobamide

Extent of conversion of ornithine to 2,4-DAP

vi

Page

102

104

106

108

110

112

114

“n

INTRODUCTION1

The initial steps in the fermentation of both ornithine and

lysine in the putrefactive anaerobe, Clostridium sticklandii, involve

 

a series of amino group migrations followed by oxidative deaminations
which prepare the amino acids for subsequent thiolytic cleaveages.
These cleavages result in the generation of acyl—CoA fragments from
which energy may be derived in the form of ATP, and in the formation
of short chain fatty acids which can be used for further biosynthetic
processes.

DL-lysine is fermented by C: sticklandii and related organisms

 

through a cleavage between carbon atoms 2 and 3 (Type A cleavage) or
carbon atoms 4 and 5 (Type B cleavage) yielding acetate, butyrate and
2 equivalents of ammonia.

6 2 1
CHSCHZCHZCOOH + CH3COOH + ZNH3

6 2 1 //f>////z
(IIHZCHZCHZCHZCIIHCOOH \

B
NH NH 6 2 1

2 2 CH3COOH + CHSCHZCHZCOOH + 2NH3

 

1A Literature Review which follows references the material
presented in this Introduction.

The first reaction in the Type A cleavage is catalyzed by a PLP- and
S-adenosylmethionine-dependent enzyme forming L—B-lysine from
L-a-lysine. L-B-lysine is next converted to 3,5-diaminohexanoate by

B-lysine mutase, an enzyme requiring a B coenzyme, ATP, a mercaptan,

12
FAD, pyruvate, and a monovalent and divalent cation for full activity.
A DPN-dependent dehydrogenase next catalyzes an oxidative deamination
forming 3-keto-S-aminohexanoate, which undergoes a thiolytic cleavage
yielding the fatty acids and ammonia.

The first and only transformation identified in the Type B
cleavage involves the migration of the e-amino group of D-a-lysine
to carbon atom S forming 2,5-diaminohexanoate. The enzyme catalyzing
this step, D-a-lysine mutase, is structurally identical to the B-lysine
mutase and has similar cofactor requirements except that PLP replaces
pyruvate and FAD is not stimulatory.

The initial reaction in the oxidation of ornithine to acetate,

NHS’ alanine and CO in C, sticklandii, proceeds through an initial

2

 

B12 coenzyme-dependent migration of the 6—amino group of ornithine to

carbon atom 4, forming 2,4-diaminopentanoic acid (2,4-DAP). This
conversion is catalyzed by the enzyme ornithine mutase. A subsequent

PLP-dependent epimerase catalyzing an inversion of the C -amino group

4
of 2,4-DAP has been proposed to exist and to precede a TPN or DPN
linked oxidative deamination forming 2-amino-4-ketopentanoic acid.
The enzyme catalyzing this‘last step (2,4—diaminopentanoic acid C4

dehydrogenase) has been partially purified and appears to have no

further cofactor requirements.

. III
I!
III

P”
OTI
d9;
COT]

Ii

 

 

man

aCiI

the

In contrast to the thoroughly studied aminomutases in the
lysine pathway, ornithine mutase has only been cursorily examined with
respect to cofactor requirements using crude or partially purified

extracts still containing 2,4-diaminopentanoic acid C dehydrogenase.

4
The purpose of the present study was to establish rigorously the
structure of the product of the ornithine mutase reaction, 2,4-DAP,
and to examine more thoroughly the properties of the enzyme catalyst.
In the course of the investigation, methods were developed for obtain-
ing gram quantities of 2,4-DAP and for separating ornithine mutase
from C4 dehydrogenase and purifying both enzymes to homogeneity. A
sensitive and rapid spectrophotometric, coupled assay employing the
pure C4 dehydrogenase was devised for measuring mutase activity. It
was demonstrated that PLP functions as a cofactor for ornithine
mutase and not in a subsequent amino group inversion as previously
suggested. In addition, the properties of both ornithine mutase and

2,4-diaminopentanoic acid C dehydrogenase have been described.

4
This thesis is organized into four sections. The first is a
literature review including a discussion of the metabolism of the

putrefactive organisms of the genus Clostridium, the fermentation of

 

ornithine and lysine in C, sticklandii, and a comparison of the 812-

dependent enzymes involved in these fermentations with related enzyme

 

systems. The other three sections consist of a published manuscript
concerning the structure of 2,4-diaminopentanoic acid, an accepted
manuscript on the purification and properties of 2,4-diaminopentanoic
acid C dehydrogenase, and a manuscript prepared for publication on

4

the purification and properties of ornithine mutase.

LITERATURE REVIEW

Part I
The Amino Acid Fermenting Clostridia

 

E i
|

The non-saccharolytic, putrefactive organisms of the genus

Clostridium are able to grow on media with amino acids as the only

 

sources of energy. These anaerobes employ certain groups of amino
acids as hydrogen (electron) donors and other groups as hydrogen
(electron) acceptors in coupled oxidation-reductions known as the
Stickland reaction (Stickland, 1934). The electron donor is oxida-
tively deaminated and thiolytically cleaved, eventually resulting in

the formation of fatty acids, C02, NH3’ ATP and DPNH. The electron

acceptor is reduced and sometimes deaminated at the expense of the

reoxidation of DPNH. In C: sporogenes, amino acids which serve as

 

efficient electron donors include alanine, valine and leucine.
Amino acids which function as electron acceptors include proline,

hydroxyproline, glycine and ornithine (Stickland, 1934; Woods, 1936).

Part II
The Fermentation of Ornithine and Lysine

Introduction. Since the early demonstration of the Stickland

 

reaction in C: sporogenes, the details of these coupled oxidation-

 

reduction fermentations have been investigated in a number of related

organisms. C: sticklandii oxidizes ornithine to acetate, NHS and

C02 (Figure l) in the presence of either proline or lysine (Stadtman,

 

1954; Stadtman and White, 1954). However, growth will not occur with
any of these amino acids alone. Dyer and Costilow (1968) and Mitruka

and Costolow (1967) showed that resting cells of C, sticklandii and

 

C: botulinum ferment L-ornithine as a single substrate. In

C. sticklandii part of the ornithine is oxidized to acetate, alanine

 

and NH:5 and the remainder reduced to 6-aminovaleric acid and NH3'
However, if proline is present with ornithine all of the d-aminovalerate
is derived from the former amino acid. In contrast, O-aminovalerate

is the main product of ornithine fermentation by resting cells of

C, botulinum. Costilow and Laycock (1971), using C, sporogenes

 

extracts, demonstrated that this transformation proceeds by an
initial conversion of ornithine to proline plus NHS’ catalyzed by
the enzyme ornithine cyclase.

In the presence of ornithine, cells of C: sticklandii degrade

 

lysine to a mole each of butyrate, acetate and NH3 (Figure l)
(Stadtman, 1963). In addition, a mole of ATP is formed from

ADP + Pi per mole of amino acid fermented. Since this conversion
occurs without a net oxidation or reduction, lysine apparently does
not serve directly as an electron donor or acceptor. However, the
lysine pathway may function as a source of reducing power with
ornithine present by generating acetyl-coenzyme A fragments,
presumably through a thiolytic cleavage (Figure l), which may

reductively condense to form butyrate (Stadtman, 1954). Alternatively,

 

.wfiecmaxumum mswvapumomu :« :oNum~:oEpow ocflmxa ecu ocazuflcuo HM izscfii

mxmchmm cofiumpcoEpow madqu

IOOU-U + ICOU-U-U-U

H N

:OOU-U-U-U + :OOU-U
o H N o

a m

_
_1_V N12 _

:2- a

:OOU-U-U-U-U-U

H

omwcomoucxcoc
:<o .m.m

2000.

a

ommuse ocdmxa-m-q

:OOU-U-U-U-U-U

H

ommuzs
ocNEm-m.N ocwmxa

N

:oou-u-u-u-u-u

H

m

:2 I2

ocwm>H-e-q

N o _

m

:2- :2-

N22 sz _

_ _
u-u-u-u-u _
N o

N N

:4 N24 ”:4 sq
:oou-u-u-u-u-u
N o a N o

omwuse
ocflmxfl-e-n

N sz sz

_ _

.:III :oou-u-u-u-u-u
N o ommEoom» N N o
ocflmxfi-c-n

 

 

ae< + :z N + tenuous + oumexosn + La + ao< + o~:N + ocamsfl

xeznuma :oflumucthew ocfizuNCLO

moo + :oou-u

N N
f

_ :Z-

:4
IOOU-U-U + 1000-0
H N m

a

_
~22 o

_ _
:oou-u-u-u-u
H N m

N

ommcomouvxcov n22-
a<a-4.~

~:z

_
:oou-u-u-u-u
A NN _ m

:2
ommeoeflmo _—

~zz ~24
:oou-u-u-u-u
A ~ m
”WGHSE
ocflcuficuo
N ~22 ~:z ~:z

:2
2000 W U U W z 00 W U U _
- - - - o - - - -u

H N m ommsoumu H N m
ocwsuflcuo-e-q ocfinuwcuo-a-o

 

N n N

:o + 00 + :2N + cumuoua N + o :v + ocwcuficco

an intermediate(s) in the breakdown of lysine, which can also be

obtained from proline, may be required as a growth factor.

Fermentation of ornithine. The oxidation of ornithine by

 

C, sticklandii to acetate, alanine and ammonia (Figure 1) involves

 

a primary cleavage between carbon atoms 3 and 4, with alanine
derived from carbon atoms 1 through 3, and acetate from carbon

atoms 4 and 5 (Dyer and Costilow, 1968). Alanine can be further
oxidized to acetate, C02 and NH3 in the presence of lysine or an
electron acceptor, proline (Stadtman, 1954; Stadtman and White, 1954;
Dyer and Costilow, 1968). The first reaction in the pathway was
discovered independently by Dyer and Costilow (1970) and Tsuda and
Friedmann (1970) and involves the migration of the 6-amino group of
ornithine to carbon atom 4, forming 2,4-diaminopentanoic acid
(2,4-DAP). The enzyme catalyzing this reaction, ornithine-4,5-
aminomutase, has been cursorily studied in crude extracts (Tsuda

and Friedmann, 1970) and appears to be cobamide coenzyme dependent.
Two further transformations proposed by Tsuda and Friedmann (1970)
result in the formation of 2-amino-4-ketopentanoic acid. They
proposed that the first of these involves the participation of a
pyridoxal phosphate dependent epimerase which catalyzes the inversion
of the C ~amino group of 2,4-DAP. A pyridine nucleotide-linked

4

dehydrogenase then oxidatively deaminates the C -amino group of

4

2,4-DAP forming 2-amino-4-ketopentanoic acid. The dehydrogenase

was partially purified and was shown to be highly specific for

fi‘h.‘m son-fl“

2,4-DAP, to utilize DPN+ or TPN+ equally well, and to have no further
cofactor requirements (Tsuda and Friedmann, 1970). The equilibrium
of the reaction is strongly in favor of oxidized pyridine nucleotide.
The existence of a pyridoxal phosphate dependent epimerase between
ornithine mutase and 2,4-diaminopentanoic acid C4 dehydrogenase was
only tentatively proposed from experiments with ammonium sulphate
treated extracts which catalyzed the reduction of DPN+ through the
overall conversion of ornithine to 2—amino-4-ketopentanoic acid. It

was suggested that a coenzyme B -dependent transformation inhibited

12
by intrinsic factor preceded a pyridoxal phosphate dependent trans—

formation which was inhibited by hydroxylamine.

Fermentation of lysine. The degradation of lysine by

 

C: sticklandii and related organisms (Figure 1) occurs either

 

through a cleavage between carbon atoms 2 and 3 (Type A cleavage)

or carbon atoms 4 and 5 (Type B cleavage) yielding acetate, butyrate
and 2 equivalents of ammonia (Stadtman, 1954, 1955). However, the
formation of acetate in cell extracts has only been demonstrated

by the former pathway (Stadtman, 1962). The initial steps in lysine
fermentation by both pathways involve a series of amino group
migrations, similar to the mutase reaction in the ornithine pathway.
These prepare the amino acids for oxidative deaminations which can
readily generate energy—rich acyl—coenzyme A intermediates through
subsequent thiolytic cleavages.

The first reaction in the type A cleavage, studied in Clostridium

 

strain SB4 extracts, is catalyzed by a pyridoxal phosphate and

S-adenosylmethionine-dependent mutase, forming L-B-lysine from
L-a-lysine (Costilow 33 31., 1966; Chirpich et 31,, 1970). The
enzyme contains bound pyridoxal phosphate and ferrous ion, is
rapidly inactivated by oxygen, and requires the presence of
mercaptans for full activity. The molecular weight of the protein
is 285,000 and it contains 6 subunits, each with 3 sulfhydryl groups.
One of the sulfhydryl groups is protected from titration with
sulfhydryl reagents in the native state (Zappia and Barker, 1970).
The next step is the type A cleavage involves the reversible con-
version of L-B-lysine to 3,5-diaminohexanate and was demonstrated

in Clostridium strain SB 93 sticklandii and Clostridium strain

4:
M—E extracts (Dekker and Barker, 1968; Tsai and Stadtman, 1968).

 

 

 

The aminomutase catalyzing this reaction is resolved during purifi-

cation from extracts of C; sticklandii into two distinct protein

 

moieties: an acidic protein with a molecular weight of 150,000 con-
taining tightly bound cobamide, and a labile sulfhydryl protein of
molecular weight 60,000 (Stadtman and Renz, 1968). In addition to

312 coenzyme, the enzyme requires ATP, a mercaptan, FAD, pyruvate,

a monovalent cation (K+, Rb+ or NH4+), and a divalent cation

(Mg+2 or Mn+2) for full activity. A DPN+-dependent dehydrogenase

next catalyzes an oxidative deamination forming 3-keto-5-aminohexanoic
acid (Rimerman and Barker, 1968), which presumably undergoes a thiolytic
cleavage yielding the fatty acid products.

The only transformation thus far identified in the type B

cleavage (Figure l) is the freely reversible migration of the e-amino

10

group of D-a-lysine to carbon atom 5, forming 2,5-diaminohexanoate
(Stadtman and Tsai, 1967). The aminomutase catalyzing this reaction,
the D-a-lysine mutase, has been detected in Clostridium strain M—E

and purified from C. sticklandii extracts (Morley and Stadtman, 1970).
The enzyme is structurally identical to the L-B—lysine mutase;

however, resolution of the complex is achieved after purification by
acidification to pH 4.0, at which point the cobamide moiety is rendered

insoluble. The sulfhydryl protein is stabilized in solution by the

FT- :31 [.'£..'~' Wfi
‘.|

addition of mercaptans. The sulfhydryl proteins of both mutases
appear to be interchangeable with the cobamide moieties directing the
specificity for either D-a-lysine or L-B-lysine (Morley and Stadtman,
1970). The cofactor requirements of the D-a-lysine mutase are
similar to those for L-B-lysine mutase except that the former enzyme
is stimulated by pyridoxal phosphate instead of pyruvate, and PAD is
stimulatory to the latter but not the former mutase. The cobamide
moietycfifthe D-a-lysine mutase contains bound pyridoxal phosphate
(Morley and Stadtman, 1972), and ATP is believed active as an allo-
steric effector (Morley and Stadtman, 1970).

Part 111
Other Coenzyme BIZ—dependent Mutases

Introduction. The ornithine, D-a—lysine, and L-B-lysine

 

mutases involved in the early transformations of ornithine and lysine

fermentation belong to a class of enzymes which utilize B coenzymes

12

as cofactors. The coenzyme forms of vitamin B are organo-cobalt

12

derivatives of the vitamins that contain a 5'—deoxyadenosine moity

11

covalently bound to the cobalt atom in place of a CN-, OH_ or H20

ligand in the corresponding vitamin. The various B coenzymes differ

12

with respect to the particular nucleotide base attached to the cobalt
on the underside of the corrin ring (see Barker, 1966, 1967, and

Hogenkamp, 1968, for comprehensive reviews of B biochemistry).

12

Clostridium sticklandii normally synthesizes the B coenzyme con-

12

taining adenine as the nucleotide base (pseudo-BIZ) (Stadtman, 1960).

 

Cobamide coenzyme (5,6-dimethy1benzimidazolyl-Co-S'-deoxyadenosylcoba-

mide), as well as pseudo-B , satisfy the B coenzyme requirement of

12 12
the mutases in the ornithine and lysine pathways (Tsuda and Friedmann,
1970; Stadtman and Renz, 1968; Morley and Stadtman, 1970). However,
the tightly bound corrinoids native to these proteins have not yet
been elucidated.

The remaining reactions catalyzed by the class of enzymes

dependent on the coenzyme forms of vitamin B have recently been

12
reviewed elsewhere (see below) and will be briefly mentioned. The

transformation common to all these coenzyme B z-dependent reactions

1
involves the replacement of a group attached to one carbon atom of
the molecule with a hydrogen from an adjacent carbon. Table 1 lists

the individual reactions catalyzed by these enzymes and Table 2

summarizes a number of properties of these proteins.

Carbon-nitrogen bond cleaving mutases (Stadtman, 1971, 1972).

 

One other enzyme catalyzing this type of transformation in addition to

the lysine and ornithine aminomutases has been described: ethanolamine

-‘—-F—"

 

1.2

. fie

ommcexcow HoNa ON: + 0:0 :u + zouu- :u

 

 

 

 

 

 

 

 

 

 

 

mfluouumm
=
= E
N N m ._u -m
awuouomm ommuvxnov Hoflo o I + oru :u :u + :0 w mu :0
:
omm>mofiu vcom :omxxo-conumu
N5 N5 :
omwuse cumuausfim = = N _
ewpouomm -ocofixnuoz-d :oou-u-:u-uoo: H :oou-u . =U-U-uoo:
_ _
m5 _ z
I
mmmeews ommuzs <ou N _
.mfiuouumm -Hx:o~ws~xzuoz <oumym-:u-uoo: H <oumww . IUIUIUOOI
o m _ o _
:u 2
N22 Nzfi I
mfiuouomm ommuse oumemusfio :00u-:u-:u-uoo: H :oou-:u -Nzu-u-uoo:
.. _ _
:u :
omm>mo~u vcom con»mu-conumu
osxucm
mo osxncm nouxfimumu cowuoaom
:oNuanNuumwo

 

 

.Aanma .cmEuvmum acumv omwcmuoou m cw vomoHuco exonm mw :owonvxn m xn

coowNQou ma umcu msouw mcfluwumwa esp :owuomoh some cm .mcofluowom «popcomovuoexncoou Nam ”H m4m<h

 

 

1.3

N22 N22 N22 2_——H—
mfiuouomm omauae ocflcpficuo Ioou Iu In :0 Io H Ioou Iu- :0 fig
I
.12 Na Na zmsm
_ N N _m I_N N _
unassumm «mange o=Lm22-a-o 2oou- 2U. 2o- 20. 2U 2U + 2ooU- 2o- N20- 2o w ~2u
I
N22 N22 N22 2 N22
N _ N _ m N _N _ N
maueuoam canons 0:2m22-m-2 2oou- 2U- 2U. 2U- 2U. 2U + 2oou- 2o- 2u-~2 2u-w- 2o
I
325.2202 m m I_N
mfiuouoam acmea2°ca2um 22 + O2u 2g + 2- u- N2u
I
omm>mofiu econ :owouuwz-=onumu
o~2 + 22-2 +
I0 I IOIO
W- W Nfl2mvm W W
A. + I
//\\_ _/,\\.: _\_ _//
u/ I Iu u I I u
ommuosvou mm-u\I// o\// \/o\
mfiuouomm oeNuooHuaconflm omen mm-uNI omen
Io .Ivflfia Io
N N _ _ _ ~ _
2:303 omeugcoc 3.3930 0 I + OIU- Io: NIU 1. :0 I0 :0

I

14

 

 

 

 

 

 

 

 

 

 

 

 

aa< .+2 . +22 mo» N N N N ooo.oNN mmaposeou
N onfluooaosconwm
fi+¢Izv+x oz H.M wconum H N ooo.wm~ ommnvxnov Nonooxfio
n+22Ufi+v222+2 oz N.M Neoaum H N ooo.oNN mmmN222oe NoNo
- mm» N.M 2mo2 N N ooo.oNN «means ouaeapsNN
ocoaxnuoaud
. mm» m New: B N ooo.mv~ ommusa opmaeusNo
- mo» N.M Naoaum N N coo.moN NmaN=<
. oz N 2mm: N N ooo.om Hmwnouomm
ommusa <00
uaxsona fixnuoz
n+2mvfl+e22v+2 oz ON.M 2mm: N 02 ooo.oom mmchsaou
ocfismaocmnum
N we» N N N N N ommusa ocfinchuo
2
5+ 222 2+:2v+2
.N+Nz .222 .2e2 mo» N.M Neonum N N ooo.oNN campus ocNmNN-a-2
v .
2+ 22VN+ezv+2 N+Nz
oum>39xm .m<m .mb< mo» N.M mconum N N ooo.oNN ommusa ocfimxfinm
confiscom omxh memo:
whouommou 2892. 230.5 Mafia ampfiWWofi manna
wcwuavom Nam venom a m H H 2
Na

.mommusz acovcomovu

m oaxucoou mo moNuuomoum "N wants

15

deaminase. This enzyme is found in an unidentified Clostridium

 

which utilizes choline and ethanolamine as substrates for growth.
The migration and exchange of the amino group and hydrogen of
ethanolamine results in the formation of an unstable intermediate

which spontaneously decomposes to acetaldehyde and NH3'

Carbon-carbon bond cleaving mutases (Barker, 1972).

 

Methylmalonyl CoA mutase catalyzes the reversible formation of
succinyl-CoA from methylmalonyl-CoA. This enzyme participates in
the oxidation of propionate, methylmalonate and compounds converted
to these acids during degradation by animal tissues and micro-
organisms, and also participates in the synthesis of propionate by
the propionibacteria. The enzyme from both animal and bacterial
sources contains cobamide coenzyme which is protected by the protein
from photolysis, in contrast to the pseudo-B12 bound to ethanolamine
deaminase, which is inactivated by visible light. With the bacterial
mutase, coenzymes containing either a purine or benzimidazole
nucleotide base can substitute for cobamide coenzyme. In contrast,
the sheep liver enzyme is specific for benzimidazole-containing

cobamides.

Glutamate mutase, found in C, tetanomorphum, Rhodospirillum

 

rubrum and R, spheroides, catalyzes the initial step in the fermenta-

 

tion of glutamate in these organisms. The protein consists of two
subunits, component E of molecular weight 128,000, and component 3 of

molecular weight 17,000. Component E binds both substrate and B12

16

coenzymes. Component S contains sulfhydryl groups which can form

both inter- and intramolecular disulfides in the presence of 02

resulting in inactivation. Component 8 apparently increases the

affinity of component E for B coenzymes. The native—bound

12

coenzyme is probably pseudo-B however, other base substituted

12;
coenzymes are equally as effective with varying affinities for the
E component.

a—methyleneglutarate mutase catalyzes the reversible conversion

 

 

of a-methyleneglutarate to B-methylitaconate. The enzyme functions in
the anaerobic degradation of nicotinic acid in C, barkeri. The B12

requirement is similar to that of glutamate mutase.

Carbon-oxygen bond cleaving mutases (Abeles, 1972).

 

Dioldehydrase isolated from Aerobactor aerogenes catalyzes the

 

conversion of 1,2-propanediol and ethylene glycol to the corresponding
aldehydes. In addition to cobamide coenzymes, coenzymes in which the
dimethylbenzimidazole moity is replaced with adenine, benzimidazole

and chloroadenine are also active. Coenzyme B12 forms an irreversible

and photolytically resistant complex with apoenzyme in the presence
or absence of substrate. However, in the absence of substrate, the

coenzyme, normally unreactive toward 0 , is converted to inactive

2

hydroxocobalamin of O is present. This fact, and the observed

2
changes in the light and ESR spectra of enzyme-bound coenzyme in the
presence of substrate, suggest that the interaction between apoenzyme

and coenzyme leads to structural changes in the coenzyme.

17

Glycerol dehydrase has been isolated from Lactobacillus,

 

Aerobacter aerogenes and a Clostridium sp. The enzyme catalyzes

 

 

the conversion of glycerol to B-hydroxypropionaldehyde, although
ethylene glycol and 1,2-propanediol also serve as substrates. The
enzyme is purified as a stable but inactive hydroxocobalamin complex
containing a large sulfhydryl protein and a smaller protein of
molecular weight 22,000. The holoenzyme is activated by the addition
of 812 coenzyme in the presence of Mg+2 and $0 =.

3

Ribonucleotide reductase from Lactobacillus leichmannii

 

 

reduces the four nucleoside triphosphates to the corresponding
2'-deoxynucleotides. The immediate physiological reducing agent
is a low molecular-weight dithiol protein which can be replaced by

Escherichia coli thioredoxin or reduced lipoic acid and related

 

dithiols. ATP apparently functions as an allosteric effector and
Mg+2 regulates the rates at which the various substrates are
reduced. The enzyme requires DBC coenzyme or an analogue containing
another benzimidazole or purine nucleotide base.

Part IV

Vitamin BIZ-dependent Reactions

In addition to the ten biochemical reactions mentioned above
which utilize cobamide coenzymes as prosthetic groups, there are
three other reactions known to require a vitamin form of B12. These
have been reviewed by Barker (1967) and Stadtman (1971), and will be

briefly mentioned.

18

Methionine synthetase catalyzes the transfer of a methyl
group from NS-tetrahydrofolate to homocysteine, forming methionine.

The enzyme is found in certain strains of E: coli, Streptococcus

 

faecalis, several other bacteria, and in the livers of animals.

Enzyme-bound Vitamin B is the intermediate carrier of the methyl

12

group in this reaction. S-adenosylmethionine is required as a

L

cofactor but apparently serves to methylate a group on the enzyme

rather than functioning in the methyl transfer reaction per se.

in

Methane synthetase, found in the methane bacteria, catalyzes
the formation of methane derived from the methyl groups of various
donor compounds. These include formate, serine, methanol, acetate,

methyl-B and NS—methyltetrahydrofolate. The corrinoid containing

12
5-hydroxybenzimidazole as the nucleotide base appears to be the
native-bound B12 vitamin. The B12 vitamin is the intermediate
carrier of the methyl group from donor molecules which is reductively
cleaved in a final step to yield methane. Reducing equivalents are
provided by molecular hydrogen and hydrogenase, or by an NADH

generating system.

Acetate synthetase is found in Clostridium thermoaceticum

 

and C, sticklandii. Methyl B functions as an intermediate methyl

 

12

carrier in the formation of acetate from C02. The reduction of C02

to protein-bound methyl B proceeds through the intermediate

12

formation of formate and NS-methyltetrahydrofolate. The acetate

synthetase-bound methyl B then undergoes methyl group carboxylation

12

in the presence of CO followed by reduction and cleavage of the

2’
carboxymethyl group to acetate at the expense of NADPH.

19

Part V

Mechanisms of BIZ-dependent Reactions

It is apparent from the discussion in Part IV that enzyme

systems dependent on a vitamin form of B are involved in methyl

12
group transformations. The formation of methionine from homocysteine
involves methyl transfer, methane formation involves methyl transfer
and methyl group reduction, and acetate synthesis involves methyl
transfer, carboxylation and carboxymethyl group reduction. The

role of 312 in these systems as a methyl group carrier has been

investigated by tracing radioactivity from 14C-methyl donors to

protein bound methyl B and then to reaction products (Barker, 1967).

12
The function of the corrinoid in the reactions catalyzed by

the coenzyme forms of B is that of the intermediate carrier of the

12
migrating hydrogen that exchanges for the group on a neighboring
carbon atom. The hydrogen is transferred to the 5'-methy1ene group
of the 5'-deoxyadenosyl moity of the coenzyme (which probably
involves rupture of the cobalt-carbon bond) forming a 5'-methyl
group (Miller and Richards, 1969; Eagar gt 31,, 1972). One of the
now equivalent hydrogens is subsequently transferred to the product.
Tritium transfer from substrate to coenzyme and from coenzyme to
product has thus far been demonstrated in the majority of the
reactions listed in Table l (Stadtman, 1971). The kinetic isotope
effects observed with tritium and deuterium labeled substrates and

coenzymes in such experiments established that the rate limiting

step in the mutase reactions is the breaking of the carbon-hydrogen

I \‘2Mum1
.- ‘

20

bond in the substrate (Abeles, 1971; Barker, 1972; Stadtman, 1972).
The role of the cobamide in the glycerol dehydrase, ornithine mutase,
and ribonucleotide reductase reactions has not yet been rigidly
established. Attempts to detect hydrogen transfer from 3H-5'-1abeled
coenzyme to deoxyribonucleotide products resulted in rapid exchange

of tritium with H20 (Beck EE.Elf’ 1966). This was postulated to occur
due to an enzyme catalyzed reversible exchange between coenzyme
hydrogen and reduced lipoate, followed by a rapid exchange of thiol
hydrogens with the solvent. No exchange has been reported to occur

between the migrating hydrogen and H20 in the other B coenzyme

12
reactions studied (Abeles, 1971; Barker, 1972; Stadtman, 1971).

It is yet uncertain as to the species of hydrogen transferred
in the mutase reactions. The appearance of unpaired electrons
during the ethanolamine deaminase reaction suggests that the hydrogen
migrates as a radical to the 5'—deoxyadenosyl moity of the coenzyme
(Babior and Gould, 1969). A mechanism involving transient cleavage
of the C-Co bond and formation of a deoxyadenosine intermediate
derived from coenzyme would appear to be plausible. If such an
intermediate exists, however, it must remain tightly bound to the
enzyme since no exchange with free added 5'-deoxyadenosine has been
detected under normal catalytic conditions (Stadtman, 1972). Such an
intermediate has been detected during the diol dehydrase reaction in
the presence of glycoaldehyde (Wagner gt gt., 1966) and during the

ethanolamine deaminase reaction with ethylene glycol as substrate

(Babior, 1970). Furthermore, 5'-deoxyinosine is liberated during the

 

21

abortive reaction of diol dehydrase in the presence of 1,2—propanediol
and deoxyinosyl cobalamin (Jayme and Richards, 1971).

In contrast to the role of B12 coenzymes as the intermediate
carrier of the migrating hydrogen, the mechanism of migration of
the constituent which replaces the abstracted hydrogen in these
reactions is open to speculation. The migration of these groups in

the various mutase reactions appears to occur intramolecularly since

neither free nor enzyme-bound intermediates have been detected. In

fawn-am”
J

addition, a variety of proposed alkyl intermediates for the reactions
involving carbon-carbon bond cleavage and rearrangement fail to be
incorporated into products (Barker, 1972). The B-lysine mutase
reaction also occurs without exchange with ammonia nitrogen from

the medium (Bray and Stadtman, 1968). Morley and Stadtman (1972)
have proposed that pyridoxal phosphate is directly involved in the
catalysis of the amino group migration in the D-a-lysine mutase
reaction. The cobamide binding protein of the enzyme complex
catalyzes a slow cobamide independent but pyridoxal phosphate-

and Mg+2-dependent exchange of the hydrogen at position 6 of D-lysine
with H20. This suggests that pyridoxal phosphate may be the carrier
of the migrating amino group through an intermediate involving
Schiff-base formation between enzyme-bound pyridoxal phosphate and
the e-amino group of the substrate. Pyruvate, the carbonyl compound
which stimulates the B-lysine mutase, may serve a similar function
in the analagous amino group migration associated with this reaction

(Stadtman and Renz, 1968).

REFERENCES

Abeles, R. H. (1971), tg_The Enzymes, Vol. 5, 3rd Edition, Boyer,
P. D., Ed., Academic Press, New York and London, p 481.

Babior, B. M. (1970), J. Biol. Chem. 245, 1755.

Babior, B., and Gould C. G. (1969), Biochem. BiOphys. Res. Commun.
34, 441.

Barker, H. A. (1966), it Symposium on B coenzymes, Barker, H. A.,

12
Ed., Fed. Proc. 25, 1623.

Barker, H. A. (1967), Biochem. J. 105, l.

Barker, H. A. (1972), tg_The Enzymes, Vol. 6, 3rd Edition, Boyer,
P. D., Ed., Academic Press, New York and London, p 509.

Beck, W. S., Abeles, R. H., and Robinson, W. G. (1966), Biochem.
Biophys. Res. Commun. 25, 421.

Bray, R. C., and Stadtman, T. C. (1968), J. Biol. Chem. 243, 381.

Chirpich, T. P., Zappia, V., Costilow, R. N., and Barker, H. A.
(1970), J. Biol. Chem. 245, 1778.

Costilow, R. N., Rochovansky, 0. M., and Barker, H. A. (1966),
J. Biol. Chem. 241, 1573.

Costilow, R. N., and Laycock, L. (1968), J. Bacteriol. 96, 1011.

Costilow, R. N., and Laycock, L. (1971), J. Biol. Chem. 246, 6655.

Dekker, E. E., and Barker, H. A. (1968), J. Biol. Chem. 243, 3232.

Dyer, J. K., and Costilow, R. N. (1968), J. Bacteriol. 96, 1617.

22

 

Jay.

Mil

Hit
M0:
M01
Rin
St:
St;
St.
St

St

St

St

St

St

St

23

Dyer, J. K., and Costilow, R. N. (1970), J. Bacteriol. 101, 77.

Eagar, R. G., Baltimore, B. C., Herbst, M. M., Barker, H. A., and
Richards, J. H. (1972), Biochemistry 11, 253.

Hogenkamp, H. P. C. (1968), Annu. Rev. Biochem. 37, 225.

Jayme, M., and Richards, J. H. (1971), Biochem. Biophys. Res. Commun.
43, 1329.

Miller, W. W., and Richards, J. H. (1969), J. Amer. Chem. Soc. 91,
1498.

Mitruka, B. M., and Costilow, R. N. (1967), J. Bacteriol. 93, 295.

Morley, C. G. D., and Stadtman, T. C. (1970), Biochemistry 9, 4890.

Morley, C. G. D., and Stadtman, T. C. (1972), Biochemistry 11, 600.

Rimerman, E. A., and Barker, H. A. (1968), J. Biol. Chem. 243, 6151.

Stadtman, T. C. (1954), J. Biol. Chem. 67, 314.

Stadtman, T. C. (1960), J. Bacteriol. 79, 904.

Stadtman, T. C. (1962), J. Biol. Chem. 237, PC2409.

Stadtman, T. C. (1963), J. Biol. Chem. 238, 2766.

Stadtman, T. C. (1972), tg_The Enzymes, Vol. 6, 3rd Edition. Boyer,
P. D., Ed., Academic Press, New York and London, p 539.

Stadtman, T. C. (1971), Science 171, 859.

Stadtman, T. C., and Renz, P. (1968), Arch. Biochem. Biophys. 125,
226.

Stadtman, T. C., and Tsai, L. (1967), Biochem. Biophys. Res. Commun.
28, 920.

Stadtman, T. C., and White, F. H. (1954), J. Bacteriol. 67, 651.

Stickland, L. H. (1934), Biochem. J. 22, 1746.

24

Tsuda, V., and Friedmann, H. C. (1970), J. Biol. Chem. 245, 5914.

Tsai, L., and Stadtman, T. C. (1968), Arch. Biochem. Biophys. 125,
210.

Wagner, 0. W., Lee, H. A., Frey, P. A., and Abeles, R. H. (1966),
J. Biol. Chem. 241, 1751. ‘

Woods, D. D. (1936), Biochem. J. 30, 1934.

Zappia, V., and Barker, H. A. (1970), Biochem. Biophys. Acta. 207,

505.

ARTICLE 1

PREPARATION AND CHARACTERIZATION OF 2,4—DIAMINOVALERIC ACID:

AN INTERMEDIATE ZUV THE ANAEROBIC OXIDATION OF ORNITHINE

BY

Ralph L. Somack, David H. Bing, and Ralph N. Costilow

Appears In:

Analytical Biochemistry, Volume 41, Number 1, May 1971

 

Copyright©197l by Academic Press, Inc.

 

 

 

IIIIIII‘

 

 

~2 >2r-«——.-—~m - _. uh...- Wu . . L - “yam-r». -a—d. :1" “Nu:- m. a.-. -

PREPARATION AND CHARACTERIZATION OF

2,4-DIAMINOVALERIC ACID:
AN INTERMEDIATE IN THE ANAEROBIC OXIDATION OF ORNITHINE

RALPH L. SOMACK, DAVID H. BING, AND RALPH N. COSTILOW

Department of Microbiology and Public Health, Michigan State University,
East Lansing, Michigan 48823

Reprinted from ANALYTICAL Brocrmurs'mr, Volume 41, Number 1, May 1971
Copyright© 1971 by Academic Press, Inc. Printed in U. S. A.

 

 

Reprinted from ANALYTICAL BIOCHEMISTRY, Volume 41, Number 1, May 1971
Copyright© 1971 by Academic Press, Inc. Printed in U. S. A.

 

ANALYTICAL BIOCHEMISTRY 41, 132-137 (1971)

Preparation and Characterization of
2,4-Diaminovaleric Acid:

An Intermediate in the Anaerobic Oxidation of Ornithine1

RALPH L. SOMACK, DAVID H. BING, AND RALPH N. COSTILOW

Department of Microbiology and Public Health, Michigan State University,
East Lansing, Michigan 48823

Received September 25, 1970

The first intermediate in the anaerobic oxidation of ornithine (2,5-
diaminovaleric acid) by Clostridium sticklandii is 2,4-diaminova1eric
acid (2,4—DAV), which accumulates in reaction mixtures containing
crude or dialyzed extracts from which cofactors are omitted (1). This
compound is formed from ornithine by a cobamide coenzyme dependent
reaction, and it is then oxidatively cleaved to form acetate, alanine, and
ammonia. The intermediate was originally identified as a dibasic com-
pound similar to ornithine by the identical electrophoretic mobilities with
ornithine at five pH values. The positions of the amino groups were
indicated by the results of oxidations with chloramine T, acid dichromate,
and periodate.

Since 2,4-DAV is a new amino acid, we decided to undertake 8 more
complete characterization. Methods are outlined for obtaining gram
quantities of the compound from reaction mixtures containing extl‘fwts
of C. sticklandii, and for preparing three derivatives. The derivatl"es
are characterized and evidence of structure is presented.

MATERIALS AND METHODS

M edia and Growth Techniques

C. sticklandii strain HF was grown in two-liter flasks in a medium
consisting of 0.6% trypticase (BBL), 0.6% yeast extract, and 06%
L-arginine-HCI in 0.04M potassium phosphate buffer (pH 7.5) under
natural gas. Cell extracts were prepared as previously described (2) 9nd
stored at ——20°C. They were not dialyzed prior to use. The lysine m'

‘JoumaI Article No. 5174, Michigan Agricultural Experiment Station.
132

/_

”—‘M—a

 

 

 

i
I
I.
I
J

 

 

2,4-DI;\.\II.\‘OVALERI(' AC“) 133

corporated in previous media (1,3) was found to contribute little to
cell growth, whereas the addition of trypticasc significantly increased
cell yields. Arginine, however, could not. be replaced by trypticasc with-
out a great loss in cell and enzyme yield. Both the total and specific
activities of extracts converting ornithine to 2,4-DAV paralleled cell
yields under these conditions. Activity increased exponentially with cell
growth and to a constant value at stationary phase.

.Alnalytical Methods

Melting points wore determined with :1 Hoover capillary melting point
apparatus. The elemental analysis was performed by Spang Micro-
analytical Laboratory, Ann Arbor, Michigan. The infrared spectrum
was obtained with a Perkin—Elmer model 700 spectrometer, the nuclear
magnetic resonance spectrum with a Varian NA—IOO MHz spectrometer,
and mass spectrometry with a LKB model 9000 combination gas chro-
matograph spectrometer. The optical rotation determination was made
with a Carl Zeiss spectropolarimetcr.

Isolation and Purification of 2,4-1)AI"

A reaction mixture of 300 ml containing 25 mM tris(hydroxymethyl)-
aminomethane (Tris)-chloridc (pH 7.5), 4 HM dimethylbenzimidazolyl-
cobamide (DBC) coenzyme. 80 1a.)! L-ornithine, 2.5 mM adenosine
diphosphate, and crude cell extract (4 gm protein estimated by the
method of Lowry cf ul. (4!) was incubated under argon in the dark
on a magnetic stirrer at 37°C for 1.5 hr. The reaction was terminated
by the addition of an equal volume of 10% trichloroacctic acid (TCA).
The protein was removed by centrifugation at 5° and 20,000!) for 10
min, and the TCA in the supernatant fluid removed by extracting three.
times with an equal volume of ether. The supernatant solution was
concentrated to a thick syrup under reduced pressure at 40° and
taken up in 20 ml of water. Ornithine. 2.4-DAV, and alanine were
separated from anions by adsorption to a Dowcx 50ll'-X4 (200-400 mesh)
H‘-f0rm column (3X21 cm). After washing the column with five,
bed volumes of water, the amino acids were eluted with 1 RI NILOH.
Ninhydrin-positive fractions were pooled, concentrated to dryness. and
brought to a 20 ml volume with chloroform/methanoI/l50? NHIOH
(40:40: 10). This solution was placed on a 6 X 35 cm silicic acid column
(SilicAR CC-4. 100-200 mesh. h‘laIlinckrodt) which had been previously
equilibrated with the above solvent. Fractions were monitored for
ninhydrin-positive compounds, and the compounds identified by thin-
Iaycr chromatography using the chloroformhncthanol05% NILOH
(36:46:20) solvent system (Table I). Alanine appeared after about 600

 

134 SUMACK, mxo, AND coerLow

TABLE 1
Chromatogimmic and ltllectrophoretic Properties of 2,4—l)iaminovaleric acid-2HCl

 

Solvent, 2,4—l)A\' [‘nknown“

 

Chromatographyh (Rf)

Butyl alcohol/acetic acid/water (60: 15:25) 0.2:»: 0.18

Propanol/pyridiue/water (1:121) 0.44 0.42

Methanol/wal-er/pyridine (20:5: 1) 0.34 0,46

Chloroform/methanol/l5‘}; NIL()H (40:40:10) 013 0.14

Chloroform/melbarrel/15‘}? NILOH (36:46:20) 066 —
Electrophoresis (cm from origin‘)

0.2 M formic acid, pH 2.0, 42.5 \'/cm, 45 min 25.40 23.40

 

" Valnas from Dyer and (,‘ostilow (1 1.

" All chromatograms were on Whatman 331M paper except. the chlorot'omi/methanol/
159:} NH40H solvents. which were on thin-layer silica gel (1 (l‘). Merck AG) plates
(40:40:10) and silicic acid TLC (ChromAR Sheets, Mallinekrodt) (36:46:20). All
systems were ascending except the butanol/acetate/water, which was descending.

" The compounds migrated as cations.

ml of this solvent was added and was completely eluted by 2000 ml. At
this point the solvent proportions were changed to 36:46:20 chloro-
form/methanol/l5"b NILOH and 2,4-DAV was eluted in the following
600 ml, after which ornithine appeared. The fractions containing 24-
DAV were pooled, concentrated to dryness, and brought to a 15 ml
volume with water. The oils accumulating from the solvents employed
were removed by ether extraction. The product was adsorbed on a Dowex
50W-X4 (100-200 mesh) Ht-form column (1.3 X 15 cm), washed, eluted
with 2 N NHDI'I, evaporated to dryness. and stored at —20°.

RESULTS: I’repm‘u/inn of .‘.4-I).4l' [brim/ices um! Eridcnce of Structure

The purified product was taken up in 10 ml of water, to which was
added 15 ml of 0.8% picrie acid in methyl alcohol. The picrate crystal-
lized on standing and was reerystallizml twice from methanol/water
(50:50), yielding 1.9 gm of monopicrate. The monopicrate was identified
spectrophotometrieal1y by the absorbance of a solution of known weight
concentration employing a molar extinction coefficient (cm) measured
for picrie acid in water of 1.25 X 10‘ .1!" cm". The monopicrate was
found to have a melting point range of 124° to 137°C, melting with
decomposition.

Crystalline 2,4-DAV tlihydrtn-hloride was prepared by adding 5 ml
of 6 N HCl to 0.5 gm of the monopicrate. The picrie acid was removed
by extraction with ether, the aqueous solution evaporated to dryness.
and the residue dissolved in 1 ml of absolute ethyl alcohol with a mini-

 

 

 

2,4-DIAMINOVALERIC ACID 135

mum amount of water. Ether was added until a permanent turbidity
developed and the suspension held at —20° overnight. The resinous
deposit. resulting was triturated with a small volume of absolute ethanol
and ether, dissolved in water, and evaporated to dryness under reduced
pressure at 40°. This deposit was then dissolved in absolute ethanol
with a minimum amount of water. Crystallization was achieved by the
addition of cold ether and the crystals were washed twice with ether.
Centrifugation rather than filtration was used to collect the crystals
since the ether-insoluble product formed a resinous deposit on paper
which was difficult to remove. The yield was 135 mg of crystals which
turned brown when stored at room temperature in closed vials. The
dihydrochloride melts with decomposition at 172° to 179°.

Elemental analysis: C5H..N2():Clg. Indicated: C 29.53, H 6.84, N
14.57, 0 16.38, Cl 32.68. Calculated: C 29.28, H 6.88, N 13.66. 0 15.60,
Cl 34.57.

The reduced ninhydrin analysis (5) of a known weight concentration
using ornithine as a standard indicated a molecular weight of 218. The
infrared spectrum (Fig. 1) revealed a broad, strong absorption at v =

 

'Yrivvr vvvvvvvvvv

% TRANSMISSION

 

 

 

 

CM"

FIG. 1. Infrared absorption spectrum of 2.4.4liaminovalerie acid dihydrochloride

(KBr pellet).

2340—3300 cm’1 (> OH and > NHf), a medium peak at v = 1840-2150
cm‘1 (> C—NHfCl“), medium peaks at v = 1635 our1 and v = 1480
cm‘1 (> NH;), and a weak absorption peak at v=1700—1735 cm"

The nuclear magnetic resonance spectrum of the dihydrochloride (Fig.
2) demonstrated the single C-2 proton by the doublet of doublets at
84.22 ppm, the C-3 methylene group by the sixteen line multiplet at
approximately 82.33 ppm, the lone C-4 proton by the two overlapping
quartets at 83.8 ppm, and the C-5 methyl by the doublet at. 81.53 ppm.
Irradiation of the proton at 83.8 ppm collapsed the methyl group to a
singlet, confirming the position of the amino group at C-4. The optical

 

,- w '19—"

:- x.»

,._V.. -_

ocuc-I ”-3.

136 soMACK, mxu, AND eosriLow

 

RELATIVE MW"

 

 

 

1%le

«A ‘40 30 20 A i “In
8 (m)

 

FIG. 2. 100 MHz nuclear magnetic resonance spectrum of 2,4-diaminovaleric acid
dihydrochloride (1)30 solution. TMS as external reference).

rotation [( or );,'-’7?, of the dihydrochloride in water (0 = 5%) was
—17.39°.

The dibenzoyl derivative was prepared by adding 0.12 ml of dibenzoyl
chloride to a cold solution of 32 mg of 2,4-DAV-2HCI in an equal
volume of 1N NaOH. The solution was acidified with HCl and the
product recrystallized twice from hot water, yielding 21.4 mg. The
melting point is 120°. Mass spectrometry gave a molecular ion of 340,
equivalent to an empirical formula of C19H20N204. The fragmentation
pattern revealed an ion of molecular weight 148, confirming the position
of the benzoyl amide on C—4.

Chromatography of a solution of the dihydrochloride supplied the R;
values in five solvent systems and the electrophoretic mobility (Table
1). The values obtained by Dyer and Costilow (1) are included for
comparison. For unknown reasons, our results with the butyl alcohol"
acetic acid/water and methanol/water/pyridine systems differ consider-
ably. Complete separation was achieved only with the chloroform"
methanol/15% NH40H (36:46:20) solvent on thin-layer ChromAR
sheets.

These results fully confirm that the. intermediate described by Dyer
and Costilow (1) is 2,4-diaminovaleric acid, and provide the necessary
characteristics for simple presumptive detection (TLC), and for identi-
fication by derivative formation and analysis by infrared and nuclear
magnetic resonance spectroscopy.

 

——--——————l'—-———"

 

I ——A‘ — ——_-—

2,4-DIAMINOVALEBIC ACID 137

SUMMARY

2,4-Diaminovaleric acid was purified in gram quantities, three deriva-
tives were crystallized and characterized, and the structure was verified
by infrared and nuclear magnetic resonance spectroscopy.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Service grants 5-ROI-
AM10791-01 and l-ROI-AMl3679-02 from the National Institute of Arthritis and
Metabolic Diseases. Ralph L. Somack is a U. S. Public Health Predoctoral Trainee,
Public Health Training Grant GM-01911 of the National Institute of General
Medical Sciences.

The mass spectrum and its interpretation were kindly performed by Professor C.
C. Sweeley and his associates at the Department of Biochemistry, Michigan State
University. We are indebted to Professor H. Hart, of the Department of Chemistry.
Michigan State University, for arranging the use of the Varian HA-100 MHz
spectrometer and to G. Love and E. Roach for their expertise in performing and
interpreting the nuclear magnetic resonance spectrum. We thank Professor J. C.
Speck for performing the optical rotation analysis, The DBC coenzyme was
generously supplied by H. A. Barker, Department of Biochemistry, University of
California, Berkeley.

REFERENCES

l. DYER, J. K., AND Cosrnow, R. N., J. Bacteriol. 101, 77 (1970).

2. Drm, J. K., AND Cos'riww. R. N., J. Bacteriol. 96, 1617 (1968).

3. STADIMAN, T. C., J. Bacterial. 67, 314 (1954).

4. LOWBY, 0. H., Roar-mauve", N. J., FARR, A. L., AND RANDALL, R. J.. J. Biol. Chem.
193, 265 (1951).

5. Moon, 8., AND STEIN, W. H., J. Biol. Chem. 211, 907 (1954).

 

 

....w

 

ARTICLE 2

2,4-DIAMINOPENTANOIC ACID C4 DEHYDROGENASE:

PURIFICATION AND PROPERTIES OF THE PROTEIN*

By

Ralph Somack and Ralph N. Costilow

To Appear In:

Journal of Biological Chemistry, December 1972

 

RALPH SOMACKT AND RALPH N. COSTILOW§

From the Department of Microbiology and Public Health,

 

Michigan State University, East Lansigg, Michigan 48823

 

SUMMARY

The NAD+-NADP+ dependent 2,4-diaminopentanoic acid C4
dehydrogenase from Clostridium sticklandii has been purified to
homogeneity by the criteria of disc gel electrophoresis and ultra-
centrifugation. The weight average molecular weight of the native
enzyme as determined by high speed sedimentation equilibrium is
72,000. Sedimentation velocity indicated an 520,w of 4.478.
Sodium dodecyl sulfate disc gel electrophoresis-and high speed
sedimentation in 6 M guanidine-HCI established that the enzyme is
composed of two subunits of identical size. The enzyme is
sensitive to thiol inhibitors and titration with S,5'-dithiobis-
2-nitrobenzoic acid and p-chloromercuribenzoate demonstrated the
lpresence of six sulfhydryl groups per mole. Amino acid analysis
indicated that the enzyme contains six half-cystine residues per

Inole.

36

37

The first two transformations in the fermentation of

ornithine to acetate, alanine and ammonia in Clostridium sticklandii

 

have previously been identified (1,2).‘ Ornithine is initially

converted to 2,4-diaminopentanoic acid in a coenzyme B 2 and possibly

1

pyridoxal phosphate dependent reaction catalyzed by ornithine mutase
(1,2). The 2,4-diaminopentanoate is then oxidatively deaminated
forming 2-amino-4-ketopentanoic acid (2). The enzyme catalyzing this

latter reaction, a C dehydrogenase, has been partially purified and

4

shown to utilize NAD+ or NADP+ as cofactors (2). This report describes

a procedure for purifying the C dehydrogenase to homogeneity. In

4
addition, determinations were made of the molecular weight of the
native enzyme and subunits, the amino acid composition, and the

sulfhydryl group content.

METHODS

General--A11 reagents were obtained from commercial sources

except for coenzyme B 1 which was provided by Dr. H. A. Barker,

12
University of California, Berkeley, and 2,4-diaminopentanoate which
was generated as described previously (3). The procedures for grow-

ing, harvesting, storing and extracting cells of Clostridium

 

sticklandii were as reported elsewhere (1). Protein concentrations

 

were measured by the method of Lowry it. a. (4).

Enzyme Assays--The activity of the C dehydrogenase was

 

4

measured by the rate of reduction of NADP+ using 2,4-diaminopentanoate
as substrate or by a coupled reaction using D-ornithine as substrate
in the presence of an excess of ornithine mutase. The former pro-
cedure was as described by Tsuda and Friedmann (2) except that

(a) NADP+ was used instead of NAD+ to eliminate problems with NADH
oxidase in extracts prior to hydroxylapatite chromatography, and

(b) no dithiothreitol was added since it was found not to stimulate
the reaction. The reaction mixtures for the coupled assay contained:

10 mM Tris buffer, pH 8.5; 5 uM B coenzyme; 20.2 uM pyridoxal

12
phosphate; 3.0 mM NADP+; 5 mM D-ornithine; 1 mM dithiothreitol; excess

levels of C4 dehydrogenase-free mutase obtained from the hydroxyl-

apatide purification step; and the C dehydrogenase in a total volume

4

38

39

of 0.5 ml. Enzyme was added after gassing the cuvettes with argon,
the mixture equilibrated to 25° and the reaction initiated by addition
of D-ornithine. The absorbance change at 340 nm in all reactions

was monitored using a Gilford model 2000 spectrophotometer. One unit
of enzyme is defined as that amount which catalyzes the formation of

l umole of NADPH per minute under assay conditions.

Disc Gel Electrophoresis--Preparative disc gel electro-

 

phoresis utilized a Canalco "Prep-Disc” apparatus with the PD-Z/lSO
lower column insert. The Ornstein and Davis (5) gel system was used
with the gel modifications employed by Gilpin and Sadoff (6). The
resolve gel was 25 mm long and the stacking gel was 10 mm long.
Both gels contained 10% glycerol (v/v). The cathode and anode
reservoir buffers consisted of 0.3 g Tris, 1.44 g glycine, 100 ml
glycerol and 0.154 g dithiothreitol per liter, pH 8.2-8.4. The
elution buffer contained 3.12 ml concentrated HCl. 28.4 g Tris,

100 m1 glycerol and 0.154 g dithiothreitol per liter, pH 8.8-8.9.
Gels were polymerized at 50 and the electrophoresis unit cooled
during operation by circulating ice water.

Analytical disc gel electrophoresis in anionic (pH 7.3) and
cationic (pH 4.3) buffer systems were performed at room temperature
with a Buchler apparatus. The gels were stained with amido Schwartz
and destained in 7% acetic acid. A modification of the sodium
dodecyl sulphate method of Weber and Osborn (7) was used for subunit
analysis. The markers used and assigned molecular weights were:

glutamate dehydrogenase, 53,000 (8); yeast alcohol dehydrogenase,

40

37,000 (9); chymotrypsinogen A, 25,700 (10); ovalbumin, 43,000 (11);

and D-amino acid oxidase, 37,000 (12).

Ultracentrifugation Studies--Dia1yzed enzyme was used for

 

studies of sedimentation velocity at 59,896 rpm at 7.30 and for high
speed equilibrium experiments. A Spinco model B ultracentrifuge with
appropriate equipment was used. The equilibrium experiments were

conducted as outlined by thantis (13).

Amino Acid and Sulfhydryl Group Analysis--After dialysis

 

against distilled water, samples containing 475 ug of protein were
hydrolyzed in 6 N HCl under vacuum at 1100 for 24 and 72 hours. The
hydrolysates were analyzed in an ultrasensitive amino acid analyzer

by the procedure of Robertson g£_§l, (l4). Tryptophan was determined
by the method of Beaven and Holiday (15) and threonine and serine
estimated by extrapolation to zero time hydrolysis. Sulfhydryl group
content was measured by titration with DTNB and p-chloromercuribenzoate

as outlined by Finlay and Adams (16).

RESULTS

Purification--A summary of the entire purification is

 

presented in Table 1. The procedures used through the hydroxylapatite
column (Step IV) were similar to those used by Tsuda and Friedmann (2)
for partial purification of this enzyme.2 However, there were some
differences in elution patterns from DEAE cellulose and hydroxyl-
apatite since we routinely used buffers at pH 7.5 while Tsuda and

Friedmann used a pH of 7.0. At the higher pH, the C dehydrogenase

4
was eluted from DEAB cellulose with 0.175 M potassium phosphate buffer,
and from hydroxylapatite with 0.025 M phosphate buffer. The dehydro-
genase was free of ornithine mutase at this point, and the mutase could
be eluted from this column by increasing the buffer strength to 0.05 M.
The C4 dehydrogenase was far from pure at this point (see
inset in Fig. 1). Final purification was achieved by using preparative
polyacrylamide gel electrophoresis. A 2-ml volume of the concentrated
eluate from the hydroxylapatite step was made 8% (w/v) with respect
to sucrose and 40 ul of a 0.01% solution of bromphenol blue was added
as an anionic marker. The sample was applied to the preparative
column which was run at a constant current of 10 milliamps. Elution
was begun immediately at the rate of 1 ml per min to insure the

removal of possible electrophoretically generated contaminants.

Fig. 1 shows the electrophoretic elution profile. A brown band was

41

42

eluted soon after the dye which was collected in 17 2-m1 fractions.
The 280 nm absorption profile of these fractions traced the C4
dehydrogenase activity. The most active fractions (No. 26-34) were
concentrated and then dialyzed for 12 hours against standard 0.1 M
potassium phosphate buffer, pH 8.5. The concentrate was stored under
liquid N2.

The specific activity of the enzyme was essentially doubled,
and only a small amount of enzyme was lost during this step. Not only
did the enzyme appear homogeneous after electrophoresis in both
anionic (pH 9.3) and cationic (pH 4.5) systems (see inset in Fig. 1),
but a single peak with no sign of heterogeneity was observed in a
high speed sedimentation velocity experiment. This was conducted
using enzyme at a concentration of 3.9 mg per ml in 0.05 M potassium
phosphate buffer, pH 7.0, containing 0.1 N KCl. The $20,w value

calculated from this experiment was 4.478.

Molecular Weight of Enzyme and Subunits--Three high speed

 

sedimentation equilibrium experiments were conducted with the purified
enzyme in 0.05 M potassium phosphate buffer containing 0.1 M KCl.‘ One
run was at pH 7.0 and sedimented for 24 hours at 25,910 rpm and 10.40
and the other two at pH 7.9 for 24 hours at 25,965 rpm and 13.90.

The plots of the ln fringe displacement against the radius squared
were linear in all cases for fringe displacements over 100 um.2

The weight average molecular weights calculated from these three

experiments were 68,260 (pH 7.0 run), 73,450 and 74,340. The average

value was 72,070.

43

Duplicate determinations of subunit structure and molecular
weight by sodium dodecyl sulphate gel electrophoresis were run on
different occasions. A single protein band was observed in all gels

containing the C dehydrogenase and the molecular weight estimated

4
from the standards employed was 40,000 i 10%. This suggests that the
enzyme has a native molecular weight of 80,000 i 10% and consists of
two polypeptide chains of equal size.

The molecular weight of the subunit was also estimated by a
high speed sedimentation equilibrium experiment with 0.6 mg/ml of
enzyme in 6 M guanidine hydrochloride containing 0.12 M mercaptoethanol
and 0.05 M potassium phosphate buffer, pH 7.7. The plot of the ln

fringe displacement versus the radius squared was linear and indicated

a subunit molecular weight of 35,380.

Amino Acid Analysis--The amino acid analysis of the C4

dehydrogenase failed to indicate anything particularly unique about

 

the enzyme. The averages of four analyses of 72-hour and two analyses
of 24-hour hydrolyzed samples indicated the following amino acid
residues per subunit of enzyme: 28 Asp, 12 Thr, 13 Ser, 32 Glu,

19 Pro, 32 Gly, 21 Ala, 27 Val, 3 1/2 Cys, 16 Met, 26 Ile, l7 Leu,

5 Tyr, 8 Phe, l7 Lys, 5 His, 9 Arg, and 22 Try.2 Calculations based
on these data indicated a molecular weight of 35,000 per subunit and

a partial specific volume of 0.73 cc g-l.

Sulfhydryl Group Content-~The C dehydrogenase was strongly

 

4
inhibited by p-chloromercuribenzoate (100% by 0.2 mM), iodoacetate

44

(98% by 2 mM) and N-ethylmaleimide (98% by 2 mN).2 Titration of
0.294 mg (4.12 mumoles) of pure enzyme with DTNB yielded a final
absorbance change of 0.309 O.D. unit after 4.5 hours indicating

5.6 sulfhydryl groups per mole of enzyme (72,000 g). Using
p-chloromercuribenzoate for titration of 2.06 mumoles of enzyme, the
final corrected absorbance change was 0.109 O.D. unit which is
equivalent to 7.0 sulfhydryl groups per mole. These results and

the amino acid analysis indicate that the C dehydrogenase contains

4
6 sulfhydryl groups (3 per subunit) and no disulfide bonds.

Absorption Spectrum--The absorption spectra of the enzyme

 

in Tris buffer at pH 8.8 and in phosphate buffer at pH 8.0 (both
buffers with 10% glycerol and 0.5 mM dithiothreitol) were identical
and typical of many proteins. There was a single absorption peak in
the ultraviolet range at 276.5 nm and none in the visible range. The

280/260 absorbance ratio was 1.33.

DISCUSSION

For the first time, a dehydrogenase catalyzing a NAD+-linked
oxidative deamination of a dibasic a-amino acid other than the
a-position has been purified to homogeneity. The existence of such an
enzyme in oxidative degradation of ornithine was first suggested by
Dyer and Costilow (18) who demonstrated that ornithine was cleaved to
form alanine from carbons 1-3 and acetate from 4 and S. Later they
showed that 2,4-diaminopentanoate was an intermediate in this degrada-
tion (1). Tsuda and Friedmann (2) subsequently confirmed the formation
of this intermediate and demonstrated the activity of a C4 dehydrogenase
in partially purified extracts. The specific activity of the dehydro-
genase preparation studied by Tsuda and Friedmann was only 1/4 of that
of our final preparation. Our data indicate that the partially
purified preparation used by Tsuda and Friedmann probably contained
many contaminating proteins. The results of the present study provide
unequivocal proof for the existence of a single protein which catalyzes
the oxidation of 2,4-diaminopentanoic acid at the 4-position with the
reduction of either NAD+ or NADP+.

Similar dehydrogenases are probably involved in the lysine
fermentation. Rimerman and Barker (19) demonstrated the activity of

3,5-diaminohexanoate C dehydrogenase. The partially purified C

3 4

dehydrogenase preparation of Tsuda and Friedmann (2) was not active on

45

46

this substrate. It appears likely that there is also a C dehydrogenase

5

for 2,5-diaminohexanoate, a product of D-a-lysine mutase (20). Intact

cells of E, sticklandii have been shown to cleave lysine at both the

 

C and C5 (21) positions but the C cleavage has not been observed in

3 5
cell extracts and the activity of such a dehydrogenase has not been
reported. Therefore, it appears unlikely that the dehydrogenase

described herein would be active on 2,5-diaminohexanoate.

10.

11.

12.

13.

REFERENCES

DYER, J. K., AND COSTILOW, R. N. (1970) J. Bacteriol., 101, 77.

 

TSUDA, Y., AND FRIEDMANN, H. D. (1970) J. Biol. Chem., 245,

 

5914.

SOMACK, R. L., BING, D. H., AND COSTILOW, R. N. (1971) Aflal.
Biochem., 41, 132.

LOWRY, O. H., ROSENBROUGH, N. J., FARR, A. L., AND RANDALL, R. J.

(1951) J. Biol. Chem., 193, 265.

 

ORNSTEIN, L., AND DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci.,

 

121, 305.

GILPIN, R. W., AND SADOFF, H. L. (1971) J. Biol. Chem., 246,

 

1475.

WEBER, K., AND OSBORN, M. (1969) J. 3161. Chem., 244, 4406.

 

ULLMAN, A., GOLDBERG, M. B., PERRIN, D., AND MONOD, J. (1968)

Biochemistry, 7, 261.

 

SUND, H. (1960) Biochem. Z., 333, 205.

 

DAYHOFF, M. 0., AND ECK, R. V. (1967-1968) Atlas of protein

 

sequence and structure, National Biomedical Research

 

Foundation, Silver Spring, Maryland.

CASTELLINO, F. J., AND BARKER, R. (1968) Biochemistry, 7, 2207.

 

-HENN, s. W., AND ACKERS, G. K. (1969) J. Biol. Chem., 244, 465.

 

YPHANTIS, D. A. (1964) Biochemistry, 3, 297.

 

47

14.

15.

16.

17.

18.

19.

20.

21.

48

ROBERTSON, D. C., HAMMERSTEDT, R. H., AND WOOD, W. A. (1971)

J. Biol. Chem., 246, 2075.

 

BEAVEN, G. H., AND HOLIDAY, E. R. (1952) Advan. Protein Chem.,

 

7, 319.

FINLAY, T. H., AND ADAMS, E. (1970) J. Biol. Chem., 245, 5248.

 

McMEEKIN, T. L., AND MARSHALL, K. (1952) Science, 116, 142.

DYER, J. K., AND COSTILOW, R. N. (1968) J. Bacteriol., 96,

 

1617.

RIMERMAN, E. A., AND BARKER, H. A. (1968) J. Biol. Chem.,

 

248, 6151.

STADTMAN, T. C., AND TSAI, L. (1967) Biochem. Biophys. Res.

 

Commun., 28, 920.

STADTMAN, T. C., AND GRANT, M. A. (1971) Methods Enzymol.,

 

17, 199.

FOOTNOTES

* This investigation was partially supported by Research Grant No. l-
ROl-A110951-01 from the National Institutes of Health. Journal

Article No. 5954, Michigan Agricultural Experiment Station.

U.S. Public Health Predoctoral Trainee, Public Health Training Grant
GM-01911 of the National Institute of General Medical Sciences.

This work was submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy. Present address: Depart-
ment of Biochemistry, University of California, Berkeley, California

94720.

To whom correspondence should be addressed.

The appreviations used are: coenzyme B dimethylbenzimidazolyl

12’

cobamide coenzyme; DTNB, 5,5'-dithiobis-2-nitrobenzoic acid.

Detailed purification procedures and results of equilibrium sedi-
mentation experiments, amino acid analysis and inhibition studies
are available as JBC Document Number , in the form of one
microfiche or 5 pages. Orders for supplementary material should
specify the title, author(s) and reference to this paper and the
JBC Document number, the form desired (microfiche or photocopy) and

the number of copies desired. Orders should be addressed to The

49

50

Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda,
Maryland, 20014, and must be accompanied by remittance to the order
of the Journal in the amount of $2.50 for microfiche or for

photoc0py, with a minimum charge of $2.50.

51

TABLE 1: Purification of C4 Dehydrogenase. Conditions of purification

and definition of units are described in the text.

 

 

 

Total . .
Enzyme Spec1f1c Fold Recovery
Step . . Activity Purification ,
“up“ (units/mg) (-fold) (6)
(units)
1. Crude extract 58,000 5.2 1 100
II. Streptomycin sulfate
+ ammonium sulfate
(40-70%), dialyzed 39,750 8.05 1.5 68.5
III. DEAE-cellulose
column 19,075 40.15 7.6 32.9
IV. Hydroxylapatite
column 8,075 152.0 28.8 13.9
V. Preparative disc gel
electrophoresis 7,125 272.5 51.5 12.3

 

 

 

 

 

52

.mhoxuwe axe ecu mo mcofluflmom any oumofiwcw
sepuon any we mend: one .ommu some aw Eouuon on new Eoum mm: :oflumawfie mo :ofiuuouflc
one .x~o>fipuommou .mEopmxm aficoflumo cam aflcoficm osu :H oechm eoflmwasm as» mo w: om

can m: om cam pumauxo page“ ecu mo aflopoum w: om wcfim: :mwonuaz: Home: wonflhumoc mm
cospomuom mm: mfimoHOLQOMuuon .flusmfip cam Haucauv Denmflm mflnp :fi umumnumsfiafl cesaoo
o>ADMHmmmpm msu Eouw aumuucmocou any mo can Aumoav :ESHou ouwpmmmfixxopw»; ecu Eonw
womhuxo page“ onu mo wwmoaocmoppuofio Ham umflw Hwofiuxamc< "womcH .flcfie Ham ovm<<v

ommcowonvxnoc v

u .o ”E: owm um oucmnuomnm .o .:0wuomum some mo H: om cam onswoo
noun :oflpmoflmflnsm ego mo >H doom 809m amends mmouxo no“: nzmvonuoz: oomv oumuumnsm
mm acficpficuo-o Mawms comemm we: oexncm .pxmu any :w amommm meow coaumufimflusm may

mo mfiflmuoa .ommcamopnxnov cu mo oHHmoum :ofiusfio capouonmonpooao How umfin o>flumuwmmwm

 

 

”H mmauHm

53

 

 

 

 

 

 

 

 

 

 

_ + .~ I1

-1 | i + -
- 1111- I + -‘
r- d
... d
P —4.--“.\‘b‘
1- d
- HBMHVW 3A0 -

111 1 1 L 1 T91

O (D N a) fi'

N. " - 0 Q

(V) BONVQHOSBV

24 32 4O 48 56 64
FRACTION NU M BER

I6

FIGURE 1.

54

Supplementary data:
a. Details of partial purification procedure.

b. High speed equilibrium centrifugation of the C dehydrogenase.

4
c. Amino acid analysis.

d. Inhibition by sulfhydryl inhibitors.

Authors:

Ralph Somack and Ralph N. Costilow

Title:

2,4-Diaminopentanoic Acid C Dehydrogenase

4
PURIFICATION AND PROPERTIES OF THE PROTEIN

SUPPLEMENTARY RESULTS

All purification steps were conducted at 0-40. Where
standard buffer is indicated, the buffer also contained 10% glycerol
(v/v) and 0.5 mM dithiothreitol. A summary of the entire purification
is presented in Table 1.

Streptomycin Sulfate Treatment--To the crude extract,
obtained as described under "Methods," an equal volume of 5% strepto-
mycin sulfate in 0.1 M potassium phosphate buffer, pH 7.5, was added
slowly with stirring. The mixture was stirred overnight and the
precipitate removed by centrifugation.

 

Ammonium Sulfate Fractionation--The streptomycin sulfate
treated extract was diluted with buffer to approximately 10 mg protein
per m1 and solid ammonium sulfate added to 40% saturation. After
stirring for 30 min, the precipitate was removed by centrifugation.
The supernatant solution was brought to 70% saturation with ammonium
sulfate, stirred for 30 min and then centrifuged. The pellet was
resuspended and dialyzed overnight against standard 0.15 M Tris
buffer, pH 7.5.

 

DEAE-cellulose Column Chromatography--A volume of dialyzed
extract containing 1000 mg of protein was placed on a DEAE-cellulose
column (1.7 x 25 cm) which had been equilibrated with standard 0.15 M
Tris buffer. The column was washed with the same buffer, followed by
0.175 M standard Tris buffer, which removed most of the protein. The
C4 dehydrogenase was then eluted by increasing the buffer strength to
0.2 M. Fractions containing activity were concentrated by ultrafiltra-
tion and dialyzed overnight against standard 5 mM potassium phosphate
buffer, pH 7.5.

 

Hydroxylapatite Column Chromatography--An aliquot of dialyzed
extract from the previous step containing 200 mg of protein was applied
to a hydroxylapatite column (4 x 12 cm) which had been equilibrated
with the buffer used for dialysis in the previous step. After thorough
washing, the C dehydrogenase was eluted with standard 0.025 M potassium
phosphate buff r. Ornithine mutase may be eluted by increasing the
buffer strength to 0.05 M. The fractions containing activity were con-
centrated by ultrafiltration to a protein concentration of approximately
10 mg per m1.

 

55

56

TABLE 1: Amino Acid Analysis of C Dehydrogenase. The data were

4
obtained from the average of four analyses from the 24-hour
and two analyses from the 72-hour hydrolyzed samples, as

described under "Methods."

 

 

Residues per .
Amino Acid 17 Leuc1ne Re51dues p2:5;3::sz
24 Hours 72 Hours

Asp 28.70 26.85 28
Thra 11.60 10.50 12
Sera 11.93 9.37 13
Glu 33.25 30.55 32
Pro 18.90 - 19
Gly 32.45 31.10 32
Ala 21.10 21.50 21
Val 26.40 27.10 27
1/2 Cys 3.08 2.61 3
Met 17.25 15.93 16
Ile 25.70 26.10 26
Leu 17.00 17.00 17
Tyr 4.72 4.88 5
Phe 7.50 8.13 8
Lys 17.35 16.70 17
His 4.46 4.84

Arg 8.95 8.13 9
Tryb - - 22

 

 

 

 

aExtrapolated to zero hour hydrolysis.

bEstimated from ultraviolet absorption in 0.1 N NaOH,
assuming five tyrosine residues per mole/2 (l6).

57

TABLE 2: Effect of Sulfhydryl Inhibitors. Assays were performed
with 2,4-diaminopentanoate as substrate as described under
"Methods" and enzyme with a specific activity of 43.7 units/
mg. The inhibitors were added at the indicated concentra-

tions before addition of enzymes.

 

 

 

. . Concentration Activity . . .
Additions (mM) (umoles NAng/ Inh1b1tlon
m1n x 10

None - 25.70 —

p-CMB 2 0 100
0.2 0 100
0.02 3.05 88.1

Iodoacetate 2 0.48 98.1
0 2 20.6 20 0

Ethylmaleimide 2 0.48 98.1
0 2 22.7 11 6

 

 

 

 

 

58

FIGURE 1: High speed equilibrium centrifugation of C dehydrogenase.

4

The initial protein concentration was 0.4 mg per ml and

 

was centrifuged at 25,965 rpm and 13.90 for 24 hours.
Details of the experiment are presented in the text. Ay,

fringe displacement.

 

 

 

59

 

 

 

 

0.5 1 1 T T I I l l r l
a”
.I
0.0- I! -
./
-()£5.. .f’ ‘—
3‘. /
s /’
c: “I C)" ‘/’. —4
"' o
./
-l.5- /o/ _.
o
-2.0- / -
’1. 1 1 1 1 1 m 1 L 1
49.5 50.0 50.5

RADIUS?- (cmzl

FIGURE 1.

ARTICLE 3

PURIFICATION AND PROPERTIES OF A PYRIDOXAL PHOSPHATE AND

BIZ-COENZYME-DEPENDENT D-a-ORNITHINE-4,S-AMINOMUTASE

By

Ralph Somack and Ralph N. Costilow

Submitted To:

Biochemistgy‘

 

61

PURIFICATION AND PROPERTIES OF A PYRIDOXAL PHOSPHATE AND

BIZ-COENZYME-DEPENDENT D-OL-ORNITHINE-4,S-AMINOMUTASE+

Ralph Somack.r and Ralph N. Costilow*

Running Title: D-a-ORNITHINE-4,S-AMINOMUTASE

+ From the Department of Microbiology and Public Health, Michigan
State University, East Lansing, Michigan 48823. Received 1972.
This investigation was supported by Research Grant No. l-ROl-
A110951-01 from the National Institutes of Health. Journal Article
No. , Michigan Agricultural Experiment Station. Ralph Somack
was a U.S. Public Health Predoctoral Trainee, Public Health Training
Grant No. GM-01911 of the National Institute of General Medical
Sciences. This work was submitted in partial fulfillment of the

requirements for the Ph.D. degree.

FOOTNOTES
Present address: Department of Biochemistry, University of
California, Berkeley, California 94720.

1 Abbreviations used are: 2,4-DAP, 2,4-diaminopentanoic acid;
coenzyme B12 (DBC coenzyme), dimethylbenzimidazolyl cobamide

coenzyme; PLP, pyridoxal phosphate; DTT, dithiothreitol.

62

ABSTRACT

The coenzyme B -dependent ornithine mutase from Clostridium

 

12

sticklandii catalyzing the conversion of ornithine to 2,4-diamino-

 

pentanoic acid (2,4-DAP) has been purified to homogeneity. A radio-
chemical assay employing 14C-labeled ornithine and a rapid, coupled
spectrophotometric assay employing 2,4—diaminopentanoic acid C4
dehydrogenase are described. Analysis by gel electrophoresis, sucrose
gradient centrifugation and SDS gel electrophoresis indicated that

the mutase has a molecular weight of about 180,000 and consists of

2 subunits of identical size. The enzyme is specific for D-a-ornithine
and is inhibited by L-a-ornithine, DL-a-lysine, and B-lysine. Kinetic
and inhibitor studies showed that ornithine mutase and C4 dehydrogenase
are directly linked and that pyridoxal phosphate is a cofactor for
ornithine mutase. The absorption spectrum of the mutase measured
directly in analytical gels indicated that a substantial amount of
native bound cobamide had been converted to inactive hydroxocobalamins.
After incubation with DBC coenzyme and subsequent dialysis, the
spectrum was more typical of bound DBC coenzyme. The ornithine mutase
reaction is reversible and proceeds to approximately an equal extent

in both directions. However, the product, 2,4-DAP, appeared to inhibit

the reverse reaction at concentrations greater than 0.7 mM when present

alone. The enzyme contains labile sulfhydryl groups and is inhibited

63

64

by oxygen. Experiments with H O-t indicated that the reaction proceeds

2
by a mechanism which excludes exchange of hydrogen with the solvent.
The initial steps in the fermentation of ornithine and lysine

in Clostridium sticklandii involve a series of amino group migrations

 

followed by oxidative deaminations which prepare the amino acids for
subsequent thiolytic cleavages and conversions to the fatty acid
products. The oxidation of ornithine to acetate, carbon dioxide and
ammonia proceeds by an initial migration of the G-amino group to
carbon atom 4 forming 2,4-DAP1 (Dyer and Costilow, 1968, 1970; Tsuda
and Friedmann, 1970; Somack §£_§}:, 1971). The accumulation of this
compound is stimulated by DBC coenzyme. A subsequent PLP-dependent

reaction involving a C amino group inversion was next proposed to

4
precede a TPN+ or DPN+ linked oxidative deamination forming 2-amino-
4-ketopentanoic acid (Tsuda and Friedmann, 1970). The enzyme catalyzing

this last step (2,4-diaminopentanoic acid C dehydrogenase) appears to

4
have no further cofactor requirements and has been purified to homo-
geneity (Somack and Costilow, 1972).

DL-Lysine is fermented by intact cells of E: sticklandii and

 

related organisms through a cleavage between carbon atoms 2 and 3

(Type A cleavage) or carbon atoms 4 and 5 (Type B cleavage) yielding
acetate, butyrate and 2 equivalents of ammonia (Stadtman, 1954, 1955).
However, the formation of acetate in cell extracts has only been
«demonstrated by the former pathway (Stadtman, 1962). The first reaction
in the type A cleavage is catalyzed by a PLP- and S-adenosylmethionine-

<dependent mutase, forming L-B-lysine from L-a-lysine (Costilow gt 31:,

6S

1966; Chirpich g£_§1,, 1970; Zappia and Barker, 1970). L-B-Lysine

is next converted to 3,5-diaminohexanoate by an aminomutase consisting
of an acidic cobamide protein and a smaller sulfhydryl protein

(Dekker and Barker, 1968; Stadtman and Renz, 1968). In addition to a
B12 coenzyme, the enzyme requires ATP, a mercapten, FAD, pyruvate,

and a monovalent and divalent cation for full activity. A DPN+-
dependent dehydrogenase next catalyzes an oxidative deamination
forming 3-keto-5-aminohexanoate (Rimerman and Barker, 1968), which
presumably undergoes thiolytic cleavage yielding the fatty acids and
ammonia.

The first reaction leading to the type B cleavage is believed
to be an initial migration of the c-amino group of D-a-lysine forming
2,5-diaminohexanoate (Stadtman and Tsai, 1967). The aminomutase
catalyzing this reaction is structurally identical to the B-lysine
mutase with similar cofactor requirements except that PLP replaces
pyruvate (Morley and Stadtman, 1970).

Since the initial demonstration of ornithine mutase activity
in crude extracts (Dyer and Costilow, 1970), the enzyme has only been
cursorily examined with respect to cofactor requirements using
partially purified extracts (Tsuda and Friedmann, 1970). The present
paper describes a method for separating ornithine mutase from the

2,4-diaminopentanoic acid C dehydrogenase and purifying the mutase

4
to homogeneity. The molecular weight and subunit composition suggests
.a.physica1 structure quite different from the cobamide-dependent

(aminomutases in the lysine pathway. In addition, evidence is presented

66

which indicates that PLP is required in the mutase reaction per se
and not in a subsequent amino group inversion as previously suggested

(Tsuda and Friedmann, 1970).

MATERIALS AND METHODS

Materials. Acrylamide and N,N'-methylbisacrylamide were

purchased from Canal Industrial Corporation. Coenzyme B d

12 an
[14C]B-L-lysine were kindly provided by Dr. H. A. Barker, Department
of Biochemistry, University of California, Berkeley. Non-radioactive

B-L-lysine and 2,4-DAP were generated with C, sticklandii crude

 

extracts and purified by the methods of Costolow gt_al: (1966) and
Somack §£_gl: (1971) respectively. The concentrations of these
diaminoacids were estimated by the reduced ninhydrin assay of Moore
and Stein (1954). L-Lysine-6-14C and DL-lysine-l-14C were obtained
from Calbiochem. DL-Ornithine-5-14C and HZO-t were products of New

England Nuclear Corporation. All other reagents were obtained from

commercial sources.

Methods. Culture and cultural methods for growing, harvest-

 

ing, storing and extracting cells of C: sticklandii (ATCC 12662) have

been reported previously (Somack §£_§l,, 1971).

Prgparative and Analytical Disc Gel Electrophoresis were

 

jperformed with a Buchler apparatus employing 6.0 mm (I.D.) diameter
glass tubing. The method of Ornstein and Davis (1964) was followed
‘with the gel modifications described by Gilpin and Sadoff (1971).

Both stacking gels (5 cm) and resolve gels (0.5 cm) contained 10% glycerol

67

68

(v/v). The buffer used in both the cathode and anode reservoirs
contained 0.3 g Tris, 1.44 g glycine and 0.154 g DTT per liter, pH
8.2-8.4. Analytical gels were stained with amido Schwartz, destained
by diffusion and stored in 7% acetic acid.

Subunit analyses were performed by a modification of the
SDS electrophoresis technique of Weber and Osborn (1969) using 5%
polyacrylamide gels. The following proteins of known molecular weight
were employed as markers: glutamate dehydrogenase (53,000), bovine
serum albumin (68,000), chymotrypsinogen A (25,700), ovalbumin (45,000),
and D-amino acid oxidate (37,000). The markers were added at a level

of 5 ug/gel.

Molecular Weight Determinations. The molecular weight was

 

estimated by the electrophoretic method of Hedrick and Smith (1968)

and by sucrose density gradient centrifugation (Martin and Ames, 1961).
The following standards were used in the electrophoretic determination:
human transferrin (74,000), beef liver catalase (24,000), alcohol
dehydrogenase (314,000), and horse ferritin (monomer) (450,000). The
standards were added to gels at a level of 50 ug/gel and the mutase

at 150 ug/gel. The relative migration of the mutase was established
from the position of the pink band in unstained gels. For the sucrose
gradient determination, 150 ug of mutase and 6 ug of lactic dehydro-
genase were layered on a 5-20% linear gradient (4.55 ml) in cellulose
nitrate tubes. The gradients were centrifuged in a SW39L rotor in a

Spinco model L preparative centrifuge at 48,000 rpm for 6.5 hr at 5°.

69

Six-drop fractions were collected and mutase activity measured by the

spectrophotometric coupled assay (below).

Enzyme Assays. Two procedures were used to assay ornithine

 

mutase: a radiochemical assay employing D-ornithine-1-14C as substrate
and a rapid and sensitive coupled spectrophotometric assay using

2,4-diaminopentanoic acid and C dehydrogenase. The C dehydrogenase

4 4

was separated from the mutase during the hydroxylapatite chromatography
step described later in this paper, and was further purified to homo-
geneity by preparative disc gel electrophoresis (Somack and Costilow,
1972). The radiochemical assay was carried out anaerobically and
protected from direct light in culture tubes as previously described
(Costilow and Laycock, 1968). The reaction mixture contained: 20 mM
D-ornithine containing 25-50 mCi DL-ornithine-S-14C; 0.1 M Tris-Cl,

pH 9.0; 10 mM DTT; 20 DM DBC coenzyme; 81 uM PLP; and from 0.015 to
0.15 units of enzyme in a total volume of 50 ul. After adding enzyme,
the tubes were gassed with argon for one min, stoppered, and incubated
for 5 min at 37°. The reaction was initiated by the addition of
substrate and terminated after 5 min with 25 ul of 0.5 M formic acid.
Precipitated protein was centrifuged out, 20 ul spotted on Chromar 500
thin layer silicic acid sheets (Mallinkrodt), and the chromatograms
developed by ascending chromatography with choloroformzmethanol:15%
NH4OH (36:46:20). Ornithine and 2,4-DAP were located by spraying with

0.01% ninhydrin in acetone. The spots were excised and the radio-

activity measured using a toluene-based scintillation fluid (Costilow

70

and Laycock, 1968). There is considerable variation between batches
of the thin layer silicic acid sheets which has resulted in deviations
reported by several investigators (personal communication from
manufacturer). We have noticed with some batches appreciable spread-
ing of radioactivity not restricted to the usual spots comprising the
amino acids. This effect is pronounced by increasing the specific
activity of the ornithine used. Therefore, it is necessary to
chromatograph a sample of unreacted ornithine-14C to make appropriate
corrections. The spectrophotometric assay was performed in the manner
described previously (Somack and Costilow, 1972) by measuring the rate
of TPN+ or DPN+ reduction at 270 using D-ornithine as substrate for

the mutase and coupling the reaction with the C dehydrogenase (Tsuda

4
and Friedmann, 1970; Somack and Costilow, 1972). One-cm light path
cuvettes contained: 50 mM sodium pyrophosphate buffer, pH 8.6;

4 mM TPN+; 1 uM DBC coenzyme; 40.4 uM PLP; 20 mM D-ornithine; 0.001-
0.002 units of ornithine mutase; and approximately 1 unit of C4
dehydrogenase in a volume of 0.25 ml. One unit of enzyme is that
amount which catalyzes the formation of l umole of 2,4-DAP per min
in the radiochemical assay or 1 umole of reduced pyridine nucleotide
per min in the coupled assay under standard assay conditions.
Specific activity is the number of units per mg of protein. When
both assays were performed simultaneously at 300 with 10X the level
of mutase in the radiochemical assay than was present in the coupled

assay, the specific activity calculated from the coupled system was

60% of that from the radiochemical system.

71

B-lysine mutase, D-a—lysine mutase and L-lysine mutase were
assayed radiochemically by the methods described by Chirpich EEHEL'
(1970), Morley and Stadtman (1970), and Stadtman and Renz (1968),
respectively. Lactic acid dehydrogenase was assayed spectrophoto-

metrically with pyruvate and DPNH as substrates.

Analytical Methods. Ornithine and 2,4-DAP in reactions

 

containing H O-t were adsorbed on small Dowex 50W-X8, H+ form columns

2
and washed with copious amounts of H20. The amino acids were eluted
with 3 N NH4OH and dried under vacuum. They were taken up in small

volumes of H20 and separated on thin layer silicic acid sheets. The
amino acid spots were excised, solubilized in 10X hydroxide of hyamine,
and assayed for tritium by scintillation spectrometry after adding a
toluene-based fluid (Costilow and Laycock, 1968).

Protein was determined by the method of Lowry §£_§l, (1951)
using crystalline bovine serum albumin as a standard. All optical
absorbance measurements were performed with a Gilford model 2000
spectrophotometer. Polyacrylamide gel scans were conducted with the
same instrument employing a Gilford linear transport attachment.
Spectra of solutions were determined with a Coleman, model 124, double

beam spectrophotometer. All radioactive measurements employed a

Packard Tri—Carb, model 3320, scintillation spectrometer.

RESULTS

Purification of Ornithine Mutase. A summary of the

 

purification is presented in Table 1. All operations were carried
out at 40 in dim light. In all chromatography steps, buffers con-

tained 10% glycerol (v/v) and 5 x 10.4 M DTT unless otherwise stated.

INITIAL STEPS. Ornithine mutase was partially purified by

 

the identical procedure developed for the purification of the next

enzyme in the pathway, the 2,4-diaminopentanoic acid C dehydrogenase

4
(Somack and Costilow, 1972). The method involves an initial strepto-
mycin sulphate treatment of crude extract, followed by ammonium
sulphate fractionation and then by DEAE-cellulose and hydroxylapatite
chromatography. The C4 dehydrogenase is eluted from the hydroxyl-
apatite column with 0.025 M potassium phosphate buffer, pH 7.5. The
column is next washed with 0.035 M buffer and ornithine mutase is
eluted by increasing the buffer strength to 0.05 M. The C4 dehydro-

genase may be further purified to homogeneity by preparative disc

gel electrophoresis (Somack and Costilow, 1972).

DISC GEL ELECTROPHORESIS. The 0.05 M potassium phosphate

 

buffer eluate from hydroxylapatite containing ornithine mutase activity
was concentrated by ultrafiltration to 4-5 mg of protein per m1.

From 50 to 75 01 containing 250 to 500 ug of protein was then layered

72

73

on individual analytical gels which were prepared and electrophoresed

in dim light as described in the "Methods” section. The electro-
phoresis unit was maintained as close to 00 with a circulating ice
water bath. With the Buchler apparatus, 12 gels containing as much

as 6 mg of total protein could be electrophoresed in a single run.
During the run, two protein bands were clearly visible, a pink band
containing mutase activity and a faster migrating yellow band. Electro-
phoresis was terminated when the yellow bands reached the ends of the
gels. The pink bands were carefully excised and macerated with a

glass Dounce homogenizer in 3.2 ml of a mixture containing: 0.1 M

3 M DTT and 10%

Tris buffer, pH 8.5; 13 DM DBC coenzyme; 2 x 10-
glycerol (v/v). The addition of DBC coenzyme to the elution buffer
significantly increased recovery. The macerate was gently stirred
at 00 under argon for 6 hr, the gel pieces removed by centrifugation,
and the supernatant solution concentrated to 1 ml by ultrafiltration.
Routinely, we recovered about 2/3 of the total mutase activity that
was placed on the gels, and observed about a 3X increase in the
specific activity after this step. Typically, about 13% of the
mutase activity observed in the crude extracts was recovered, and
the specific activity of the purified enzyme was about 70X that of
the crude preparations (Table l).

The mutase appeared homogeneous by the criterion of disc
gel electrophoresis. Single discrete bands deve10ped on both 7% and

5% gels electrophoresed at pH 9.3. Figure 1 shows a scan of a 7% gel

containing the pure mutase superimposed on a scan of a 7% gel

 

74

electrophoresed with enzyme from step 4 (hydroxylapatite) of
purification. The faster migrating large peak in the latter gel
is the yellow band mentioned above. The pure mutase was found to
be free of D-a-lysine mutase, L-a-ornithine mutase and B-lysine
mutase activity. (Enzyme, 8.5 pg per reaction, with a specific

activity of 1.8 units per mg, was used in these assays.)

Stability. The highly purified mutase lost 35% of its
activity in one month when stored in 50 ul aliquots at -200.
Enzyme from step 4 of purification lost 30% of its activity after
two days at 4°. By including 10% and 20% glycerol (v/v), the loss
at 40 was reduced to 20% and 9%, respectively. The addition of
20 uM DBC coenzyme also reduced the loss at 40 to 9%. The inclusion
of D- or L-ornithine or PLP in the buffer had no effect on stability.
The protection afforded by glycerol and DBC coenzyme was also observed
during attempts to purify the mutase on Sepharose-6-B columns. A
4-fold increase in recovery was achieved by the addition of 10%

glycerol and 2 uM DBC coenzyme to the buffer.

Molecular Weight and Subunits. The molecular weight of the

 

mutase from step 4 of purification was estimated by the gel electro-
phoresis technique of Hedrick and Smith (1968) and by sucrose density
gradient centrifugation. The sucrose gradient (Figure 2) utilizing
lactic acid dehydrogenase as a reference standard indicated a $20,w
value of 8.75 which corresponds to a molecular weight in the order of

170,000. The molecular weight estimated by the Hedrick-Smith technique

indicated a value of 175,000-180,000.

75

The number and molecular weight of subunits was investigated
by SDS gel electrophoresis. In two experiments using as reference
standards either ovalbumin, D-amino acid oxidase, and chymotrypsinogen
A or glutamate dehydrogenase, ovalbumin and bovine serum albumin, one
major band with a molecular weight of 95,000—98,000 and a minor
component with a molecular weight of approximately 83,000 was observed.
A scan of the mutase gel (Figure 3) shows that the minor component
represented only approximately 2% of the total protein and therefore
probably was a breakdown product or minor contaminant rather than a
subunit of the enzyme. These data and the results obtained with the
intact enzyme indicate that ornithine mutase has a molecular weight of

about 180,000 and consists of 2 polypeptide chains of equal size.

Temperature, pH Optimum, and Protein Concentration. The

 

optimum temperature for the mutase reaction under standard radio-
chemical assay conditions was approximately 370, with one-half maximal
activities occurring at 230 and 490 (Figure 4). Other experiments
demonstrated that the mutase is rapidly inactivated at temperatures
exceeding 45°.

The optimum pH was found to be 9 with one-half maximal
activities occurring at approximately 7.4 and 9.7. The pH curve
(Figure 5) indicates that potassium phosphate buffer is inhibitory.

We also observed that the addition of potassium ion to reactions in

Tris buffer decreased activity.

76

With the radiochemical assay, a plot of activity versus
protein concentration was linear in the range of 10 ug to 90 ug of
protein per reaction and extrapolated through the origin. Enzyme
from step 4 of purification with a specific activity of 1.2 units per

mg was used in this experiment.

Substrate Specificity and Anaiggues. The enzyme from
DEAE-cellulose columns utilized either 0- or L-ornithine as substrate.
However, following hydroxylapatite treatment (Step 4), no activity
was detectable with L-ornithine as substrate when tested with either
assay. Therefore, D-ornithine is the true substrate for the ornithine
mutase reaction and it is likely that an ornithine racemase was
removed by step 4 of purification.

The apparent Km for D-ornithine obtained by the radiochemical
assay was 6.7 x 10-3 M (Figure 6). The Km observed using the coupled
assay was 4.4 . 10-4 M. This large difference in observed Km values
may have resulted from the great differences in the protein concentra-
tions utilized in the two assay systems and/or the differences in
temperature and buffers used. It was not possible to determine the
Km by both procedures under the same conditions because of differences
in the sensitivity of the assays and the optimum conditions for the
C4 dehydrogenase activity. Table 2 summarizes the effect of a number
of amino acids and compounds structurally related to ornithine when
equimolar amounts were added with D-ornithine. It is of interest to

note that the presence of L-ornithine and the substrates of the

77

D-a-lysine mutase, L-a-lysine mutase and the B-lysine mutase
significantly decreased activity. Tsuda and Friedmann (1970)
reported that L-a-lysine did not act as an inhibitor when tested

using a partially purified extract.

Pyridoxal Phosphate Requirement. The highly purified mutase

 

from step 5 of purification was almost completely dependent on PLP.

The deletion of this cofactor from the radiochemical assay mixture

(8.5 ug of pure mutase, specific activity of 1.8 units per mg)

resulted in an 85% loss of activity, and the addition of either
isoniazid or hydroxylamine at a concentration of 20 mM completely
inhibited the reaction. These results provide evidence for enzyme
bound PLP or a PLP analog. PLP was previously shown to stimulate

the conversion of ornithine to 2-amino-4—ketopentanoic acid in ammonium
sulphate fractionated extracts containing both ornithine mutase and C4
dehydrogenase (Tsuda and Friedmann, 1970). However, it was proposed
that PLP functioned as a cofactor in a reaction following ornithine

mutase which catalyzed the inversion of the C -amino group of 2,4-DAP

4
prior to the oxidative deamination by the C4 dehydrogenase. The Km
for PLP reported by Tsuda and Friedmann using a partially purified
6

extract containing both mutase and C dehydrogenase was 1.3 x 10- M.

4

The apparent Km which we obtained with pure ornithine mutase employing

the coupled assay and pure C dehydrogenase was 3.6 x 10.7 M (Figure 7).

4

The value obtained using the radiochemical assay was 1.8 x 10-6 M.

Again, the differences in assay conditions may have resulted in the

78

differences in the apparent Km observed. Further evidence that the
two enzymes are directly linked is provided in Figure 8. It can be
seen that the product generated by pure ornithine mutase served as a

substrate for the pure C dehydrogenase. The initial rate of TPN+

4
reduction was directly correlated with the amount of 2,4-DAP produced
by the mutase. There was no reduction of TPN+ in the absence of

either enzyme with D-ornithine as substrate.

Absorption Spectra of the Enzyme. The marked stimulation of
mutase activity in crude or partially purified extracts by DBC coenzyme,
and the inhibition resulting from the addition of intrinsic factor

previously established the participation of a B coenzyme (Dyer and

12
Costilow, 1970; Tsuda and Friedmann, 1970). In the present study,
very little activity was detected in the absence of DBC coenzyme with
enzyme preparations following the hydroxylapatite step of purification.
The addition of DBC coenzyme to the extraction buffer to stabilize

the mutase during the final step of purification (step 5) precluded
the spectral investigation of the nature of the native bound corrinoid.
Therefore, a spectrum was derived directly from the pink band con-
stituting ornithine mutase activity in gels electrophoresed with
enzyme from step 4 of purification. This was accomplished with a
linear transport gel scanner attached to the Gilford spectrophotometer
as described in detail in Figure 9. The spectrum in the UV region

shows an absorbance maximum at 280 nm and a 280/260 ratio of 1.34.

The spectrum in the visible region is typical of a corrinoid compound

79

with a peak at 355-360 nm and a smaller absorption at 520-540 nm.

The former peak indicates that a substantial amount of bound coenzyme

is in the hydroxocobalamin form. The spectrum of pure enzyme from

step 5 of purification, dialyzed to remove the DBC coenzyme added to
stabilize the enzyme, is shown in the same figure. The broad absorp-
tion band in the visible region with shoulders in the neighborhood of
355 nm and 520-530 nm and the high absorbance in the 260 nm region is
indicative of the DBC coenzyme. Even after exposure at 40 to a 100 watt
tungston filament lamp at 12 cm for 4 hr, the spectrum was essentially

unchanged.

Extent of Conversion of Ornithine to 2,4-DAP. The conversion
of ornithine to 2,4-DAP approached 45% using either enzyme from step 4
of purification or pure mutase from step 5. Equilibrium was usually
attained within 40 min employing D-ornithine-14C at a concentration of
20 mM. Attempts to measure the extent of the reverse reaction using
similar levels of 2,4-DAP-14C resulted in little, if any, accumulation
of ornithine. Performing assays with much lower concentrations of
product to test for possible inhibition would have required excessively
high specific activity radioactive 2,4-DAP. Therefore, the extent of
the reverse reaction using low levels of 2,4-DAP was estimated by
employing the C4 dehydrogenase reaction modified as a substrate assay
(see legend of Figure 10). Levels of 2,4-DAP in excess of approxi-
mately 0.7 mM led to progressively less utilization. The extent of

conversion in both directions was measured in the manner indicated

80

above employing 20 mM D-ornithine or 0.7 mM 2,4-DAP. The results
presented in Figure 10 demonstrate that the reaction is readily
reversible and proceeds to approximately the same extent in both

directions.

Essential Sulfhydryl Groups and Effect of Oxygen. The

 

activity of the mutase is markedly decreased in the presence of

0.2 mM p-chloromercuribenzoate or N-ethylmaleimide and moderately
affected by iodoacetate at a level of 2 mM (Table 3). This suggests
that ornithine mutase contains sulfhydryl groups essential for activity,

a feature common to the majority of coenzyme B -dependent mutases

12
catalyzing carbon chain rearrangements and those catalyzing amino
group migrations. The failure of arsenite to cause inhibition suggests
that the essential sulfhydryl groups are not vicinal.

The effect of DTT and oxygen are presented in Table 4.
Similar to the three mutases in the lysine pathway, ornithine mutase
exhibited little activity under aerobic conditions either in the
presence or absence of a mercaptan. However, in contrast to the former
enzymes, about 75% of maximal activity was recovered in the absence
of DTT if oxygen was depleted by gassing with argon. DTT completely
replaced the anaerobic requirement achieved with argon. Presumably,

the mercaptan functions to keep essential, labile sulfhydryl groups

reduced and protected from oxygen.

Hydrogen Exchange. A standard reaction employing pure

 

ornithine mutase (8.5 ug, specific activity of 2 units per mg) was

81

carried out in the presence of a level of H O-t providing a total of

2
5 x 108 cpm or 9 x 106 cpm per uatom of hydrogen. The reaction

mixture was incubated for one hr and the diamino acids separated and
analyzed for the incorporation of tritium as described under "Methods."
There was no incorporation of tritium into non-exchangeable positions
of either 2,4-DAP or ornithine. An identical reaction employing
D-ornithine-14C as substrate indicated the formation of 0.12 umoles of
2,4-DAP. If exchange had occurred to the extent of one uatom of
hydrogen per umole of substrate converted and there was no isotope

selection, approximately 3.2 x 105 cpm would have been detected in the

2,4-DAP analyzed.

DISCUSSION

The conversion of ornithine to 2,4-DAP catalyzed by ornithine
mutase has been previously studied only in crude (Dyer and Costilow,
1970) or partially purified extracts (Tsuda and Friedmann, 1970) of

C, sticklandii. In the present paper, a method is described for

 

separating ornithine mutase from 2,4-diaminopentanoic acid C dehydro-

4

genase and obtaining the mutase as a pure protein. The C4 dehydro-
genase may be further purified to homogeneity by an additional
preparative disc gel electrophoresis step (Somack and Costilow, 1972)
and employed in a sensitive spectrophotometric assay to measure

mutase activity. The addition of DBC coenzyme to the buffer used to
extract the mutase from individual analytical disc gel slices signifi-
cantly increases the yield in the final purification step. This could
explain the failure of numerous initial attempts at final purification

using preparative gel electrophoresis columns where the B compound

12
was not employed in elution buffers. As much as 2 mg of pure enzyme
may be obtained in a single run by the present procedure using the
Buchler analytical system with large (6 mm I.D.) diameter glass tubing.
The ornithine mutase appeared to be homogeneous by the
criterion of disc gel electrophoresis in 5% and 7% anionic gels. On

SDS gels, however, a small amount of material constituting 2% or less

of the total protein migrated slightly ahead of the dissociated mutase

82

83

and probably represented a breakdown product of the mutase or a minor
contaminant. The 70-fold purification achieved indicates that the
mutase accounts for about 1.5% of the total soluble protein in crude
extracts.

Molecular weight estimations using sucrose gradient centri-
fugation and gel electrophoresis combined with SDS gel electrophoresis
indicate that the enzyme has a molecular weight of approximately
180,000 and is composed of 2 subunits of identical size. The ornithine
mutase appears to be strikingly different from the two enzymes in the
lysine pathway catalyzing analogous amino group migrations. The
B-lysine mutase (Stadtman and Renz, 1968) and the D-a-lysine mutase
(Morley and Stadtman, 1970) are physically indistinguishable and exist
as complexes of two distinct protein moieties, an acidic protein with
a molecular weight of 150,000 containing tightly bound cobamide, and
a labile sulfhydryl protein of molecular weight 60,000. The two pro-
teins of the B-lysine mutase are resolved during purification but
resolution of the D-a-lysine mutase has been achieved only after
acidification to pH 4.0 where the cobamide protein is rendered
insoluble. When crude dialyzed extracts containing ornithine mutase
were treated with acid at pH 5.0, the activity recovered (50%) was
found only in the resulting precipitate.

D-a-Ornithine has been identified as the true substrate for
the ornithine mutase reaction since L-ornithine fails to serve as
substrate for the purified enzyme. The L-isomer is inhibitory. It

is likely that an ornithine racemase was removed by purification step 4

84

since either D- or L-ornithine is used as substrate by more crude
enzyme preparations.

The evidence presented strongly argues against the proposal
by Tsuda and Friedmann (1970) that a PLP-dependent epimerase catalyzing
the inversion of the C4 amino group of 2,4-DAP follows the ornithine
mutase reaction in the conversion of ornithine to 2-amino-4-keto-
pentanoic acid. The data in Figure 8 show that only two pure proteins,
ornithine mutase and C4 dehydrogenase, are required for this conversion.
Furthermore, the almost complete dependence of the mutase on PLP with
a very low apparent Km (2 x 10.6 M to 4 x 10‘7 M) and the complete
inhibition of activity by isoniazid and hydroxylamine, indicate that
PLP functions as a cofactor for ornithine mutase. The possibility of

contamination of the highly purified mutase by a C epimerase is

4
further discounted by the complete lack of hydrogen-exchange noted
during the mutase reaction. Highly purified preparations of D-a-
lysine mutase are contaminated by a PLP-dependent lysine racemase
(Morley and Stadtman, 1972) which catalyzes the incorporation of
hydrogen from water into the a-position of both isomers of lysine.
The ornithine mutase has a number of features in common
with the lysine aminomutases. Thus, ornithine mutase, D-a-lysine
mutase and L-a-lysine mutase are PLP dependent whereas pyruvate may
serve the function of PLP in the B-lysine mutase reaction. Morley
and Stadtman (1972) have demonstrated that the cobamide moiety of

the D-a-lysine mutase complex contains bound PLP and in the presence

of Mg+2 catalyzes a PLP dependent exchange of a C6 methylene hydrogen

85

of D-lysine with the solvent. This suggests that PLP may be the
carrier involved in these amino group migrations through enzyme
directed PLP-substrate amino group Schiff base intermediates.

Like the D-a-lysine and B-lysine mutases, purified ornithine
mutase contains tightly bound cobamide or degradation products thereof
and activity is stimulated by the addition of DBC coenzyme. The
native bound coenzyme is nevertheless readily exchangeable with DBC
coenzyme which subsequently is resistant to photolysis. Since free
DBC coenzyme is readily inactivated by light, bound coenzyme is
presumably protected in such a way as to prevent the photolytic
reaction. This result is reminiscent of the binding of a benzimidazole-
containing cobamide coenzyme to sheep methyl-malonyl coenzyme A mutase
(Cannata g£_§l:, 1965)._ The indication that a substantial amount of
native bound cobamide appears in the hydroxocobalamin form after
purification (Figure 9) suggests that either a photolytically
susceptible bound cobamide other than DBC coenzyme is the native

prosthetic group (C: sticklandii synthesizes adenyl-cobamide coenzyme,

 

Stadtman, 1960), or a significant amount of hydroxocobalamin from
photolytic decomposition in extracts is subsequently bound to the
enzyme.

Presumably, the cobamide functions as the carrier of the
ornithine C4 hydrogen which exchanges for the amino group on C5.
This hydrogen carrier function has been demonstrated in the analagous
L-B-lysine mutase (Retey §£_§l,, 1969), the D-a-lysine mutase (Morley

and Stadtman, 1971), and a number of other B -coenzyme dependent

12

86

reactions (Abeles, 1971; Barker, 1972). In all cases, the hydrogen
migration occurs without exchange with solvent hydrogen. This is
apparently also true for the ornithine mutase reaction, since tritium
uptake could not be detected when the reaction was carried out in the
presence of HZO-t. However, the possibility of isotope selection has
not been eliminated.

Ornithine mutase contains sulfhydryl groups which must be
kept reduced either by the exclusion of oxygen or the addition of a
mercaptan. This is a common feature of the lysine aminomutases which
exhibit little activity in the absence of added mercaptans (Stadtman
and Renz, 1968; Morley and Stadtman, 1970; Chirpich g£_§l,, 1970).

The D-a-lysine mutase is inhibited by a number of diamino-
acids including L-B-lysine, 3,5-diaminohexanoate, and L-ornithine
(Morley and Stadtman, 1970). It is interesting to note that the
substrates of the lysine aminomutases, D-a-lysine, L-a-lysine and
B-lysine also inhibit ornithine mutase.

The precise equilibrium positions of the D-a-lysine and
B-lysine mutase reactions are unknown. Both proceed in the forward
direction to the extent of approximately 45-50% and in the reverse
direction to about 25-30% (Stadtman, 1972). The formation of addi-
tion products between pyruvate and 3,5-diaminohexanoate probably
inhibit the B-lysine mutase reaction in the reverse direction (Stadt-
man and Renz, 1968). With respect to the ornithine mutase reaction,

the inhibition observed in the reverse direction with 2,4-DAP concen-

trations in excess of 0.7 mM is undoubtedly a different phenomenon

87

unless adduct formation with PLP prevents the cofactor from participation

in the reaction.

REFERENCES

Abeles, R. H. (1971), ip_The Enzymes, Vol. 5, 3rd Edition, Boyer,
P. 0., Ed., Academic Press, New York and London, p 481.

Barker, H. A. (1972), iE_The Enzymes, Vol. 6, 3rd Edition, Boyer,
P. 0., Ed., Academic Press, New York and London, p 509.

Chirpich, T. P., Zappia, V., Costilow, R. N., and Barker, H. A.
(1970), J. Biol. Chem. 245, 1778.

Cannata, J. J. B., Focesi, A., Mazumder, R., Warner, R. C., and
Ochoa, S. (1965), J. Biol. Chem. 240, 3249.

Costilow, R. N., Rochovansky, O. M., and Barker, H. A. (1966),
J. Biol. Chem. 241, 1573.

Costilow, R. N., and Laycock, L. (1968), J. Bacteriol. 96, 1011.

Dekker, E. E., and Barker, H. A. (1968), J. Biol. Chem. 243, 3232.

Dyer, J. K., and Costilow, R. N. (1968), J. Bacteriol. 96, 1617.

Dyer, J. K., and Costilow, R. N. (1970), J. Bacteriol. 101, 77.

Gilpin, R. W., and Sadoff, H. L. (1971), J. Biol. Chem. 246, 1475.

Hedrick, J. L., and Smith, A. J. (1968), Arch. Biochem. Biophys.
126, 155.

Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J.
(1951), J. Biol. Chem. 193, 265.

Martin, R. G., and Ames, B. N. (1961), J. Biol. Chem. 236, 1372.

Moore, S., and Stein, w. H. (1954), J. Biol. Chem. 211, 907.

88

89

Morley, C. G. D., and Stadtman, T. C. (1970), Biochemistry 9, 4890.

Morley, C. G. D., and Stadtman, T. C. (1971), Biochemistry 10, 2325.

Morley, C. G. D., and Stadtman, T. C. (1972), Biochemistry 11, 600.

Ornstein, L., and Davis, B. J. (1964), Ann. N. Y. Acad. Sci. 121,
305.

Retey, J., Kunz, F., Stadtman, T. C., and Arigoni, D. (1969),
Experientia 25, 801.

Rimerman, E. A., and Barker, H. A. (1968), J. Biol. Chem. 243,
6151.

Somack, R. L., Bing. D. H., and Costilow, R. N. (1971), Anal.
Biochem. 41, 132.

Somack, R., and Costilow, R. N. (1972), J. Biol. Chem. (ip_p£g§§).

Stadtman, T. C. (1954), J. Biol. Chem. 67, 314.

Stadtman, T. C. (1955), ip_A. Symposium On Amino Acid Metabolism,
McElroy, W. D., and Glass, H. B., Eds., Baltimore, Johns
Hopkins University Press, p 493.

Stadtman, T. C. (1960), J. Bacteriol. 79, 904.

Stadtman, T. C. (1962), J. Biol. Chem. 237, PC2409.

Stadtman, T. C. (1972), ip_The Enzymes, Vol. 6, 3rd Edition, Boyer,
P. D., Ed., Academic Press, New York and London, p 539.

Stadtman, T. C., and Renz, P. (1968), Arch. Biochem. Biophys. 125,
226.

Stadtman, T. C., and Tsai, L. (1967), Biochem. Biophys. Res. Commun.
28, 920.

Tsuda, Y., and Friedmann, H. C. (1970), J. Biol. Chem. 245, 5914.

90

Weber, K., and Osborn, M. (1969), J. Biol. Chem. 244, 4406.
Zappia, V., and Barker, H. A. (1970), Biochem. Biophys. Acta 207,

505.

 

91

 

 

 

TABLE 1: Purification of Ornithine Mutase.a
Total Specific Fold
.5525: “77::77'
. (units/mg)
(units) (‘fOldl
I. Crude extract 61.5 0.061 1 100
II. Streptomycin sulphate
+ ammonium sulphate
(40-70%), dialyzed 44 0.077 1.25 80
III. DEAR-cellulose column 25 0.37 4.8 40
IV. Hydroxylapatite
column 11.8 1.51 24.5 19.3
V. Disc gel electro-
phoresis 7.8 4.38 71 12.8

 

 

 

 

 

aConditions of purification are outlined in the text.

Activity was measured by the radiochemical assay as described under

"Methods."

92

TABLE 2: Effect of Compounds Structurally Related to Ornithine

on Mutase Activity.a

 

 

 

Activity
Amino Acid Added (cpm in Inhibition

standard (%)

assay)
None 1100 -
L-a-Ornithine 464 57
L-a-Lysine 799 26
DL-a-Lysine 413 62
B-Lysine 477 56
N-a-Acetyl-L-ornithine 1100 0
Putrescine 1140 0
6-Amino—n-valerate 1000 9
Cadaverine 1200 0

 

 

 

aThe standard radiochemical assay was employed with
8.5 ug of pure enzyme. All compounds were present at a concen-

tration of 20 mM.

 

TABLE 3: Effect of Sulfhydryl Inhibitors on Mutase Activity.a

 

 

 

Additions Concigigation (ufigtgvgtgbz) Inhfigstion
None - 32.2 -
p-CMB 0.2 3.9 88

0.02 22.5 30
Ethylmaleimide 0.2 11.0 66
0.02 31.0 4
Iodoacetate 2.0 20.3 37
0.2 25.7 20
Arsenite 2.0 33.5 0

 

 

 

 

aRadiochemical assays were performed as described under

"Methods." Reactions contained 15 ug of protein from step 4 of

purification which was dialyzed against 100 mM Tris-Cl (pH 8.0)

containing 10% glycerol (v/v) to remove DTT. The inhibitors were

added at the indicated concentrations before the addition of enzyme.

 

94

 

 

 

TABLE 4: Effect of Dithiothreitol and Oxygen on Mutase Activity.a
Additionb TreatmentC (udifgxstTO ) % of Control (1)
(1) DTT Argon 21.1 -

(2) - Argon 16.2 77

(3) DTT - 21.2 101

(4) - - 4.0 19

(5) DTT 02 9.4 45

(6) - 02 3.0 14

(7) DTT 02 + argon 19.9 94

(8) _ 02 + argon 9.2 44

 

 

 

 

aThe enzyme preparation free of DTT used for the experiments

reported in Table 3 and the standard radiochemical assay were

employed.

to enzyme at a final concentration of 10 mM.

bWhen present, the mercaptan was added to reactions prior

CGasing with oxygen

(1 min) and/or argon (2 min) was initiated after the addition of

enzyme.

 

FIGURE 1:

95

Disc gel electrophoresis of pure ornithine mutase.
Analytical disc gel electrophoresis was performed and gels
scanned in electrophoresis buffer at 650 nm as described
under "Methods." Protein from step 4 of purification

(44 mg) was electrophoresed on one gel (----) and 14 ug of

).

 

pure mutase on the other (

96

 

 

|.5/( . /(

A |.0 P -
E
c
o s
8 0.8 - f: -
(J) :3
Z 0 6 7- 7-
% ..
(I o
8 . ‘5 ..
CO 0.4 - . 5. E
4 : ' o

: ..- >4

' :7

 

 
   

 

 

  

 

 

TOP

FIGURE 1.

GEL LENGTH (cm)

4 BOTTO

.ncfis Ham ovm<<1v mom .mov “Dwayne ocwcuwcho may

.Amxmmmm macho paw maflmuop Hmpcoeflhomxo how :mpocuoz: oomv m.m mm .mflhh z H.o was

Bee 2 mioH mcficflmpcoo HE mH.o mo oesflo> a :fi ommcowoupxnop pace afluomH mo m: 0 use

:ofiumofimfiusm mo 4 meow scum :wououm mo w: oom AHA: ponoxmfi we: mHE mm.vv uncapMHm

amonosm pmocfifi womnm < .ammuss ocfispfizuo mo :ofiumwsmwnunoo ucoflpMHw xufimcop amouozm

 

”N mmDUHm

Ii. 1 .I ll

98

(V) EONVBHOSBV

 

 

1
£0

ZOI x NIW/HNdl SB'IOW‘N'

34
TOP

  
 
 
 
 
 
 

I8 20
FRACTION NUMBER

l6

l4

 

I
OH, 12
BOTTOM

FIGURE 2.

l 1 J L l
V N

AllAllOV 38‘!an

 

99

FIGURE 3: SDS gel electrophoresis of pure ornithine mutase. Ten ug
of pure mutase were applied to the gel which was electro-

phoresed at 200 as described under "Methods."

 

100

 

 

a _ _ q _ —
o mEoEooRU

V

 

_ _ _ _ _

:5. 82 3243084 ”122.9.

J

I

 

5 BOTTOM

4

GEL LENGTH (cm)

TOP

FIGURE 3.

101

.mwe pom was: o.Hv cowumuwmwnsm mo 4 meow Eonw Dwayne mo mm mm

pocflmucoo mcowuomom .DHSHmHomEou :H cofiumflhm> can now umooxo Manama unspcmum

ecu :fi wouospcou one: mxammm O>wuomowpmm

 

.ommuss ocflnuficno mo aseflumo OHSHMHOQEOH

”v mmDUHm

 

102

 

 

1

l J l

 

 

0.

T
to 0 Q’ N
o' o’ o' o‘
All/\llOV OlleBdS

25 30 35 40 45 50 55

20

TEMPERATURE,°C

FIGURE 4.

 

103

.mopSuxHE :ofluOMOH Hoppcoo .m: pofiwom scum panammoe

mm: mm Macaw one .mmE you mafia: v.Hv cofiumofiwfiusm mo 4 zoom Scum camponm mo mm mm

nag: wouusvcoo who: wxmmmm Hw0flEo:00fipmu pumpnmum

 

.omMpsa ocwnpflcno mo Edafiumo In

”m mmDOHm

 

104

.m mmaon

O. m m h w m

 

   

265.562-8630 . 1
sad .655 o
2~.o.x-oeofimoeo . ..

s. _.o.oz-26£o o

 

Nd

v.0

0.0

md

0..

N._

v._

 

 

All/\liOV OldIOBdS

105

.me pom mafia: w.N
mo xow>fiuom afimwoomm m cum: ensues onwcuwcao when we w: m.w pocfimueou :owuomon
comm .HO>OH ocwcuwcaoio cw cowumwnm> ago now umooxo poonmEO mm: semmm HNOHEOno

uofipMH phmpcmum one .mmmuss ocwnuwcuo Dunn spa: ocflnufi:H01o How uofim xnsmuuo>mozocfiq

 

no mmDOHm

 

106

To. x..ze_..Tz_ztzmo..£
m N _ O

 

 

_ d _ _

 

 

 

.o mmDuHm

(_\_l CO V
,_(N1wxdv0 SEI'IOW‘N')

(O

O
N

107

.fima pom mafia: w.m .xufl>wuum uflmfioommv Dwayne ocwzuw:ho chum mo m: mw.o pocfimucou
macapumom .HO>OH man a“ :oHuMwam> may now umooxo poxofimso we: semmm pofimsoo pnwpcmum

one .ommuss mcwcuwauo anon spa: oumcmmocm meopflnxm How uoflm xHSm1Ho>mozocwq

 

”n mmDUHm

 

108

.5 mmDUHm

721772-585 ..<xoo.m>&

0. m m v N O N1 V1
‘ i q a _ _ _ ,

 

1
V.
o

l
0°.
0

1
‘9

l 1
Q N
N —
9-01 x,_(N1w/HNdl SB'IOWTI)

 

l
“I
N

 

 

 

 

FIGURE 8:

 

109

Conversion of ornithine to 2-amino-4-ketopentanoic acid

by mutase and C dehydrogenase. The standard radiochemical

4
assay mixture with 8.5 ug of pure mutase (specific activity,
4.3 units per mg) was used. Aliquots of 20 ul were removed
from the reaction mixture at the indicated times, diluted
1:1 with distilled H20, boiled for 30 seconds, and centri-
fuged. Volumes of 20 ul were then chromatographed on
silicic acid to assay for 2,4-DAP as described under
"Methods" (0). Volumes of 5 ul were employed in the C4

dehydrogenase assay with pure dehydrogenase and the initial

rate of TPN+ reduction recorded (O).

 

cccccc

110

(o—o) 201 x NlW/HNdl SB‘IOWTI

 

 

 

 

(I) (D Q’ N
I I 1 1
O

1- O

—- O

,— s

-- o
1 1 1 1 n 1
O I". 0. ‘0. 0. ‘0.
m’ N N - - o

1!

(H1201 x dVO-b‘Z 93‘1ow

IS 20 25 30
MINUTES

l0

FIGURE 8 .

 

111

 

.xcmfin a me Momma; mflmxfim«v Ono :pflz

moupo>20 Nuhmsc Eu H mcwm: pocfishouop mm: Amucflom among“: mafia oomnuv sapwoomm one
.Hux z H.o wcwcfimucoo .o.m mm .nommsn oumnmmonm esfimmmuom 2 mo.o umcwmwm pouxfimfip was
”ma non mafia: m.m .HE won me n.ov Dwayne Dunn Eduuuomm Hague any you .mov Ham xcman
was campoum mo oucmppomnm :mozuon camcofio>m3 some on mucouomwflp any Eonm wo>finop

ma sapwoomm use .cfiouOHm now: pawoponaouuoofio How ago :a pawn amends xnfim any

:0 mcfimSOOM Mauve pmumommn paw Mommas mflmonosmonu00Ho cw flow xcefin may no mcwmsuow
An msumammmm upommcmnu HBOCMH whomfiwu on» mcwm: some one: mnuwcoHa>m3 ceasefipafl

any we mmcwpmom :.mp0:ua£: Hope: poafiuomop we a; m.m mom campoam o: mcwcflmacoo How a
no“: pomononmouuuofio one How opfiemfixnowxfiom Hmowuxflmcm Nu a on pOflHmmm mm: “aflououm

mo w: ommv aofiumofimfiasm mo 6 doom aoum pomnuxm .opflemnou pczonnommusa you oocopfl>m

 

um mmDOHm

112

.m mmDUHm

A85 Ik02w4m><>> .

 

 

_.o
No
no ..
«.0
m .o
6.0
so

(H) BONVBHOSBV

 

0mm 0N0 00¢
.

00v ovm 0mm
_

q . d _ _ 4

   

   

      

        

 

co. m. v.6!
O O OO

)BONVBHOSBV

 

NO.

 

8:

 

I

.ommpss 0: use mcoflumuucoocoo msoHam> um a<o1v.m mcficfimucoo
appuxfie :ofiuomou fichucoo a scam pouospumcou o>uao pumpcmum m on o>fium~on coflpOSpog
+zmh mo Dump Hmfluwcfl on“ Eonm paymefiumo we: umoH no poumyoeom m<ouv.~ omwpcoomom

och .ommcowoppxcop vu mung mo mafia: moo.o no“: sammm pOHmsoo any mo mucocomeou on»

113

mcficwmwcoo mapuo>su ou poppm pcm .AAMmmoooc mm pBOSHfip .paaflon one: muoscflfim moefip
pOHMOAch ecu u< .nov a<ouv.m 25 n.o pew oexuco whom mo m: m.w no .nov ocfinuficnoio
25 ON paw Ame hog mafia: o.m .xp«>flpum onfiuommv Dwayne anon mo m: 5H spa: poxofimso

we: Ammmm Hmoflaonooflpmn pumpcmum one .d<auv.m ow ocflguflcuo mo :oflmno>:ou mo uaouxm

 

 

”OH mmDOHm

114

 

I I I
O
:5
o
l l I
80 I00 I20

I
O
l
0
MINUTES

 

 

 

 

 

L_1l=:—' : l l l
O O O O O
O (D to Q' N

CII-OV OIONVlNEdONIWVIO-V'Z °/o

FIGURE 10.