ACT|ONS INSTRUMENTAL {N THE NEW RE. N HEXOSES 'METABOUSM OF COMMO Thesis {or Jfl'ua Degree 0! Ph; D. ahfilC‘fElGAN STATE UNNERSHY Mamziwh Yeh‘m Kama! 196-5 L I BRA R Y Michigan State Univcm'ty This is to certify that the thesis entitled New Reactions Instrumental In The Metabolism of Common Hexoses presented by Mamdouh Yehia Kamel has been accepted towards fulfillment of the requirements for m._ degree in _B:Lonh.emis t ry Major professor Date OCtOber 15, 1965 0-169 ABSTRACT NEW REACTIONS INSTRUMENTAL IN THE METABOLISM OF COMMON HEXOSES by Mamdouh Yehia Kamel This thesis defines the enzymic basis for the initia» tion of the metabolism of common hexoses in Aerobacter aerogenes PRL—R3. This organism could not be shown to possess kinases for D—mannose, D—fructose, or D—mannitol even though it could utilize these compounds constitutive— ly as sole carbon sources. Its constitutive hexokinase was purified over lOOO-fold from extracts and shown to be highly stereospecific for D-glucose. The kinase was characterized with respect to pH optimum, substrate specific city, metal ion specificity, Michaelis constants, inhibition constants, and stability. The product of the D—glucokinase~ catalyzed reaction was identified as D-glucose 6-phosphate. An apparent D-mannokinase activity was detected in crude cell extracts, but was shown actually to involve an apparent 2—epimerization of D—mannose to D—glucose, the latter of which could be phosphorylated with ATP by the stereospecific D-glucokinase. The apparent 2—epimerization .k-snwmg. r Mamdouh Yehia Kamel was‘resolved into a cyclic process involVing D—mannose 6-phosphate isomerase, D—glucose 6-phosphate isomcrase D-glucokinase, and a new constitutive phosphotransfera e which could phosphorylate D-mannose with D—glucose 6-phosphate, acetyl phosphate, or carbamyl phosphate, but not with adenOSine triphosphatec The new phosphotransferase was purified several hundred fold and characterized with respect to pH optimum, phosphoryl donor specificity and kinetic constants, phOSphoryl acceptor specificity and kinetic constants, inhibition constants, stability, and reverSi— bility of the catalyzed reactions. The reaction products were prepared and identified. The Significance of the enzyme in metabolism was discussed. NEW REACTIONS INSTRUMENTAL IN THE METABOLISM OF COMMON HEXOSES By Mamdouh Yehia Kamel Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1965 ACKNOWLEDGMENT I wish to express my sincere appreciation to Dr. R. L. Anderson for his encouragement, guidance and constructive criticism throughout the course of this work. I am also grateful to Mrs, Verachtert for technical aSSistance during a portion of this research and to Mr. D. P. Allison for his co-operation and help in the preparation of the coupling enzymes. Thanks go to Mr. T. Hanson for his help in developing the sugar phosphate separation.method, to Mr. J. W. Mayo for the preparation of L—glucose and L-fruc“ tose, and to Mr, R. R. Hart for the preparation of L—galac— tose. I greatly appreciate the financial support from the Egyptian government. Words cannot express the depth of my gratitude to my wife Samira for her patience, understanding and encourage— ment during the course of my graduate work. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . vii VITA. . . . . . . . . . . . . . . . . x INTRODUCTION . . . . . . . . . . . . . . l . g PART I. PURIFICATION AND PROPERTIES OF THE STEREOe SPECIFIC D- GLUCOKINASE OF AEROBACTER AEROGENES PRL— R— 3. . . . . 3 Experimental Procedure 3 Results 6 Purification of D—glucokinase. 6 PrOperties of D-glucokinase 9 pH Optima . 9 PhOSphoryl donor specificity C Metal ion specificity 9 Substrate specificity 17 Product inhibition 28 Stability 35 Product identification . 35 Effect of growth substrate on D—gltl<::o- kinase level . . . 35 Discussion 36 Summary of Part I. 38 PART II. A CYCLIC PATHWAY FOR THE METABOLISM OF D-MANNOSE . . . . . . . 39 Experimental Procedure LO Results and discussion 42 Whole cell fermentation. A2 Absence of D—mannokinase 42 iii PART III. Apparent 2— —epimerization of D—Mannose to D—Glucose Inactivation of the D- Mannose to D; Glucose Conversion by Charcoal Inhibition of the D—Mannose to D— Glucose Conversion by Alkaline Phosphatase . ATP— —Dependence of the D Mannose to De Glucose Conversion Evidence that D— Glucose was not Derived from D— Glucose Phosphate by the Hydrolytic Action of a Phosphatase. . Measurement of the Conversion of D— Mannose to D—Glucose 6—Phosphate in a Continuous Spectrophotometric Assay; Discovery of Acyl Phosphate:Hexose Phosphotransferase . Fractionation of the Enzymes Involved in Acetyl Phosphate—Dependent and ATP— Dependent Conversion of D—Mannose to D— Glucose 6— —Phosphate . Hexose 6- -Phosphate: Hexose 6— Phosphotrans- ferase Reconstitution of the Reactions Involved in the Apparent 2— Epimerization of D— Mannos e to D— Glucose . A Cyclic Pathway for the Metabolism of D— Mannos e Summary of Part II PURIFICATION AND PROPERTIES OF ACYL PHOS— PHATE: HEXOSE PHOSPHOTRANSFERASE (HEXOSE PHOSPHATE: HEXCSE PHOSPHOTRANSFERASE) FROM AEROBACTER AEROGENES PRL— —R3 Experimental procedure. Results. , . . . Purification of Acyl PhosphatezHexose Phcsphctransferas e Properties of Phosphotransferase and Product Identification. . . pH Optimum. . . . . . Phosphoryl Donor SpeCifiCity. . Phosphoryl Acceptor Specificity. Identification of the Products of Phosphorylation of D-glucose, D— Mannose, D—Fructose, and D—Mannitol with Acetyl Phosphate . . iv A8 56 57 57 61 61 63 7O 73 80 81 82 BA 8A 89 89 95 Page Evidence for the Common Identity of Acyl PhosphatezHexose Phosphov transferase and Hexose phosphate: Hexose Phosphotransferase . . . llO Phosphatase Activity of Phosphotransv ferase and Reversibility of the D-Mannose 6—PhosphatezDeGlucose 6a Phosphotransferase Reaction . . 111 Effect of Growth Substrate on Phosphotransferase Level . . . 123 Stability of Phosphotransferase . . 127 Discussion . . . . . . . . . . . 128 Summary of Part III . . . . . . . . 131 REFERENCES. . . . . . . a . . . . . . . 133 Table I. II. III. IV. VI. VII. VIII. IX. LIST OF TABLES Page Purification of D—Glucokinase . . . . . . 10 Phosphoryl donor Specificity of D-glucokinase. 13 Metal ion specificity of D—glucokinase . . . 16 Effect of added alkaline phosphatase on the formation of D— glucose from D—mannose by a crude cell extract. . . . . . . . 58 Demonstration of hexose phosphatezhexose phosphotransferase activity: formation of D—glucose 6—phosphate from D-glucose plus D-mannose 6-phosphate. . . . . . . . 68 Reconstitution of the D-mannose to D-glucose conversion with purified enzymes . . . . 7A Purification of acyl phosphate: hexose phospho— transferase . . . . . 92 Phosphoryl donor specificity of Phospho— transferase . . . - 95 Stiochiometry of the D—mannose 6—phosphate: D— —glucose 6— —phosphotransferase reaction, and D— —glucose 6- -phosphatase activity of phosphotransferase. . . . . . 126 vi LIST OF FIGURES 1. pH Optima of D—glucokinase 2. Lineweaver—Burk plot relating D—glucokinase reaction velocity to ATP concentration 3. Lineweaver-Burk plot shoWing the relationship of D—glucose concentration to D-glucokinase reaction velocity in the presence of various contractions of D—glucosamine A. Lineweaver-Burk plot show1ng the relationship of D—glucose concentration of D—glucokinase reaction velocity in the presence of various concentrations of D—xylose 5. Kinetic plot for obtaining the K1 for D— glucosamine . . . . . . . 6. Kinetic plot for obtaining the Kl for D— xylose. . . . . . . 7. Specificity of D-glucokinase 8. Lineweaver- Burk plot relating D— —giucokinase reaction velocity to D— —g1ucose concentra— tion . . . . . . 9. Lineweaver—Burk plot showing the relationship of ATP concentration t: D—gitcckinase reac— tion velocity in the presence of various concentrations of ADP. . . . . . . 10. Kinetic plot for obtaining the K1 for ADP ll. Fermentation of D-glucose, D- -manncse and D- fructose by cells of A aipgoenes PRL¢R3 grown on D— —g1ucose mineral salt and on peptone—beef extract l2. Titrimetric assay for D—glucokinase and D- mannokinase in a crude extract and an ammonium sulfate fraction vii Page 12 15 19 21 23 25 27 30 AA 47 _—————— v, --,— -0 sup-3&- qr . 34". Figure 13. 1A. 15. l6. 17. 18. 19. 20. 21. 22. 23. 2A. 25. 26. Titrimetric assay for D—mannokinase type activity in a crude extract Double reciprocal plot of the rate of D«glucose formation as a function of D—mannose concen- tration in a crude extract Formation of luC-D-glucose from lL‘C—D-«mannose in a crude extract Inactivation of the ability of a crude extract to convert D—mannose to D—glucose by chroma— tography on Sephadex G~25 and reactivation with ATP . . . . . . . . . . . Measurement of the D-glucose formed from D— mannose and from Hexose phOSphates in a crude extract. Chromatography of a crude extract on Sephadex G_750 o e e e s o e o e o e e 0 Proposed reaction sequence for the conversion of D-mannose to D—glucose in crude extracts. Summary of the reactions believed to be instrumental in the metabolism of D-mannose in A. aerogenes PRL-R3. Typical elution profile of phosphotransferase on Sephadex G- 200 . . . . . . . . . Elution profile of phosphotransferase on DEAE cellulose . . . . . . . . pH Optimum of acyl phosphate: hexose phospho— transferase . . . . . . Phosphoryl donor kinetic constants for phosphotransferase Lineweaver-Burk plot relating phosphotrans- ferase reaction velocity to substrate cone centration. . . Lineweaver-Burk plot relating phosphotrans~ ferase reaction velocity to D—mannitol concentration. viii Page 50 52 55 6O 63 67 72 76 8'8 91 9A 98 100 102 Figure 27. 28. 29. 30. 31. 32. 33- 34. Chromatography of the products of phosphory- lation of D-glucose, D—mannose, and D- fructose with acetyl phosphate . . . . Chromatography of the product of phosphory— lation of D—fructose with acetylo phosphate. Lineweaver-Burk plot showing the relationship of D—glucose concentration to phosphotrans- ferase reaction velocity in the presence of various concentrations of D-mannose . . Kinetic plot for obtaining the K1 for D- mannose using either D-mannose 6—phosphate or acetyl phosphate as the phosphoryl donor. Lineweaver—Burk plot showing the relationship of D-glucose concentration to reaction velocity at constant concentrations of acetyl phosphate and D-mannose 6—phosphate Competitive phosphorylation of D-glucose with D-mannose 6—phosphate and acetyl phosphate Phosphotransferase- catalyzed disappearance of D- -glucose 6- -phosphate in the presence and absence of D— —mannose . . . Formation of l“C-D—mannose 6—phosphate from D— glucose 6—phosphate and 1 C—D—mannose. o Page 107 109 113 115 117 119 122 125 VITA Mamdouh Yehia Kamel was born in Cairo, Egypt on June 29, 1933. He graduated from Beni-Suef High School in June 1950 and received the B.S. degree in Chemistry and Botany from Cairo University in June, 1954. He accepted a scholarship from the National Research Center in Cairo, Egypt in 1954 and received the M.S. degree from Cairo University in 1957-1958. In 1958 he accepted a predoctoral fellowship from the Egyptian Government for further graduate work in Moscow University in Russia. In 1960 he changed his place of study to the United States of America and was accepted as a doctoral candidate in the Department of Biochemistry at Michigan State University in the summer of 1961. He will receive the Ph.D. degree in the fall of 1965. Mr. Kamel is married and has a daughter, Hebba. , _ "'"u, a.» - INTRODUCTION In organisms in which carbohydrate metabolism has been thoroughly investigated, the degradation of D- mannose is initiated by phosphorylation at carbon atom 6 with ATP in a reaction mediated by a nonspecific hexokinase. D-Mannose 6-phosphate is then isomerized by a specific enzyme to yield D-fructose 6—phosphate, which may be metabolized further via the Embden-Meyer— hof pathway or, after isomerization to D-glucose 6- phosphate, via one of the hexose monophosphate pathways. Hexose and pentose utilization in Aerobacter aero— genes PRL-R3 has been shown to proceed through reactions of both the Embden-Meyerhof pathway and a hexose mono- phosphate (transketolase-transaldolase) pathway (1-4). Observations in this laboratory, however, had indicated that the initiation of the metabolism of hexoses in this organism did not conform to the established patterns. Its constitutive hexokinase appeared to be stereospecific for D-glucose; attempts to demonstrate unequivocally the existence of D-mannokinase in this organism had.consis- tently yielded negative results in spite of the fact that both D-mannose and D-glucose could be utilized constitutive- 1y as sole carbon sources. Consequently, an investigation of the enzymic mechanisms involved in the initiation of the metabolism of D-mannose and D-glucose in A; aerogenes PRL-R3 became the subject of this thesis. In addition, some observations on the metabolism of D—mannitol and D-fructose are reported. This thesis consiSts of three parts. Part I describes the purification and properties of D—glucokinase from A; aerogenes PRL-R3 and establishes its unique stereospecifi— city. Part II describes a novel cyclic pathway for the metabolism of D-mannose which is independent of the involve— ment of D-mannokinase. Part III describes the purification and properties of a unique phosphotransferase, which is a key enzyme in the pathway described in Part II. Two abstracts and a preliminary communication on aspects of this work have been published (5-7). PART I Purification and Properties of the Stereospecific D-Glucokinase of Aerobacter aerogenes PRL—R3. As noted in the general introduction, there was an indication that D—glucose but not D—mannose could be phosphorylated with ATP in A. aerogenes PRL—R3, although either of these hexoses could be metabolized constitutively as a sole carbon source. This implied (i) that the constitutive hexokinase of this organism had a unique specificity, and (ii) that D-mannose was metabolized by an unknown mechanism. To establish these points, the enzyme which catalyzed the phosphorylation of D—glucose with ATP was purified and its properties investigated. This section of the thesis describes the stereospecific D-glucokinase (ATP:D—g1ucose 6—phosphotoransferase, EC 2.7.1.2) of 5. aerogenes PRL—R3, presents a procedure for its purification over 1,000-fold, and establishes its reaction product as D—glucose 6-phosphate. EXPERIMENTAL PROCEDURE Growth of Cells— A- aerogenes PRL-R3 was grown in 100— liter volumes in a New Brunswick Model 130 Fermacell fer- mentor at 30° with an aeration rate of 6 to 8 cubic feet per minute and an agitation speed of 300 rpm. The medium consisted of 1.35% Na2HPOu.7H20, 0.15% KHzPou, 0.3% (NHu)2 SO“, 0.02% MgSOu.7H20, 0.0005% FeSOu.7H20, 0.02% Dow Corning Antifoam B, and 0.5% D-glucose (autoclaved separ— ately). The inoculum was 2.5 liters of an overnight culture in the same medium minus the antifoam. The cells were harvested with a Sharples AS-l2 centrifuge 8 to 9 hours after inoculation. The yield was about 10 g (wet weight) of cells per liter. Chemicals— L-Galactose was prepared by R.R. Hart by borohydride and sodium amalgam reduction of D—galactur— onic acid (8). L-mannose was prepared by nitromethane addition to L-arabinose (9). L—glucose was prepared by J.W. Mayo by modifications of the procedures described by Hudson (10) and Frush and Isbell (ll). L—Ribulose and D— and L-xylulose were prepared by refluxing L—arabinose and D— and L-xylose, respectively, with pyridine (12) and were purified by chromatography on Dowex l—borate (13) after removing excess aldopentose by crystallization. L- Fructose was prepared enzymically by J.W. Mayo by an un- published procedure. D—Allose and D-altrose were gifts of Dr. F. J. Simpson. D—Mannose 6—phosphate isomerase and D-glucose 6—phosphate isomerase were purified from extracts of A. aerogenes PRL-R3 by an unpublished procedure developed in this laboratory. Phosphoglucomutase, glucose 6—phosphate dehydrogenase, lactic dehydrogenase (containing pyruvate kinase), and all other chemicals were obtained from commercial sources. D—Mannose and D—galactose were re— crystallized (14,15) before use to remove interfering amounts of D—glucose. D-Glucose 6—phosphate was determined spectrophoto— metrically by measuring the 340 mu absorhance in the presence of NADP and D-glucose 6—phosphate dehydrogenase. D-Mannose 6—phosphate was determined spectrophotometri— cally with these same reagents with the addition of D— mannose 6-phosphate isomerase and D-glucose 6-phosphate isomerase. D-Glucokinase Assay- D—Glucokinase was routinely assayed by measuring NADP reduction at 340 mu with a Gilford absorbance-recording spectrophotometer.thermo— stated at 25° using microcuvettes With a l-cm light path. The reaction mixture contained in a volume of 0.15 ml: 10 umoles of glycylglycine buffer (pH 7.5), 0.5 pmole of ATP, 1.0 umole of MgClz, 0.1 umole of NADP, 5.0 pmoles of D-glucose, excess glucose 6—phosphate dehydrogenase, and D-glucokinase at concentrations which gave a linear reSponse. The reaction was initiated by the addition of D-glucokinase. The activity of 6-phosphogluconate dehydro— genase (measured by replacing D-glucose plus ATP with 6—pthphogluconate in the assay mixture) in the crude cell' extract was always less than 20% of the D-glubokinase activity and, therefore, was not considered to contribute significantly to the observed D—glucokinase rate. Protein was determined spectrophoto- metrically with the aid of a nomograph (courtesy of Calbiochem) based on the data of Warburg and Christian (16). A unit of enzyme was defined as the amount which catalyzed the phosphorylation of l umole of D—glucose per hour under the conditions described. Unless stated otherwise, the reported experiments were performed with the most highly purified fraction of D-glucokinase. An alternate method for measuring kinase activity (as in the specificity experiment described in Fig. 7) was a pyruvic kinaserlactic dehydrogenase—linked assay based on the continuous spectrophotometric measurement of ADP (13). RESULTS Purification of D—Glucokinase All operations were performed at O to 4°. Extracts were prepared by disrupting cells of A. aerogenes PRL—R3 suspended in water in a Raytheon lO-kc sonic oscillator. The broken-cell suspension was centrifuged at 13,200 x g, and the resulting supernatant solution was used as the cell extract. The extract used in the purification des— cribed below was obtained from 700 g (wet weight) of cells. Bentonite Fractionation— Powdered bentonite, 225 g, was suSpended in 3,350 m1 of cell extract containing 54 mg of protein per ml with a 280:260 mp ratio of 0.69. Removal of the bentonite by centrifugation yielded a supernatant (2,300 ml) of 7-fold purified D—glucokinase containing 6.6 mg of protein per ml with a 280:260 mu ratio of 0.61. First Ammonium Sulfate Fractionation— Ammonium sulfate, 30.3 g, was dissolved in the above fraction, followed by 100 ml of 7.6% protamine sulfate. The preCipitate that formed was removed by centrifugation and discarded. To the super— natant solution (2,375 ml) was added 1,169 g of ammonium sulfate (80% of saturation), and the resulting preCipitate was dissolved in water to give 142 ml of 22-fold purified D-glucokinase containing 22 mg of protein per ml Wlth a 280:260 mu ratio of l 15. Acid Precipitation— The above fraction was diluted to 600 ml with water and the pH was lowered to 4.4 by the addition of acetic acid. The preCipitated protein was removed by centrifugation and discarded. The pH of the supernatant solution was immediately raised to 7.0 with ammonium hydroxide. This yielded 600 m1 of 33rfold purified D-glucokinase containing 3.8 mg of protein per ml with a 280:260 mu ratio of 1.12. Second Ammonium Sulfate Fractionation- To the above fraction was added 550 m1 of saturated ammonium sulfate (pH 7.0). The precipitate of crystalline.and-amorphous protein which appeared was removed by centrifugation and and discarded. To the supernatant solution was added 400 m1 of saturated ammonium sulfate (pH 7.0). The resul- ting precipitate was collected by centrifugation and dissolved in water to yield 16 ml of 78—fold purified D-glucokinase containing 28 mg of protein per ml with a 280:260 mu ratio of 1.18. Sephadex G-100 Chromatography- The above fraction was placed on a column (5 x 153 cm) of Sephadex G-100.equi1ib- rated with 0.01 M sodium phOSphate buffer (pH 6.5) and eluted with the same buffer. Twenty-m1 fractions were collected, and those which contained most of the activity were pooled. This yielded 120 m1 of 590-fold purified D-glucokinase with a protein concentration of 0.33 mg per ml and a 280:260 mu ratio of 1.55. DEAE-Cellulose Chromatography- DEAE-Cellulose (Bio- Rad Cellex D, exchange capacity = 0.95 meg per-g) was pretreated as recommended by Peterson and Sober.(17) and equilibrated with 0.01 M sodium phosphate buffer.(pH 6.5) in a column 1.5 x 12 cm. The above fraction was added to the column and eluted with 500 m1 (5 m1 fractions) of the same buffer containing NaCl in a linear gradient from 0 to 0.8M. The five fractions containing most of the activity (6.6% of the D-glucokinase activity of the cell extract) were 1,530- to 1,980—fold purified, contained 0.20 to 0.35 mg of protein per ml, and had 280:260 mu ratios ranging from 1.61 to 1.71. A summary of the puri- fication procedure is given in Table 1. Properties of D-Glucokinase pH Optima- D-Glucokinase activity as a function of pH was maximal at pH 7.5 in glycylglycine buffer and at about pH 8.9 in glyCine buffer (Fig. 1). Phosphoryl Donor Specificity— The relative rates of D-glucose phosphorylation in the presence of various phosphoryl donors (3.3mM) is given in Table II. ATP was the most effective phosphoryl donor. The observed phos- phorylation with ITP was competitive with ATP, the rate with 3.3 mM ATP being 34% inhibited in the presence of 13.2 mM ITP. With saturating (33.3mM) D-glucose, the Km for ATP was determined to be 0.8 mM (Fig 2). Metal Ion Specificity- After treatment of purified D-glucokinase with 0.01 M EDTA (pH 7.0) and removal of excess EDTA by passage through a Sephadex column, activity was nil in the absence of added divalent cations. The relative rates of D—glucose phosphorylation by the Sephadex- treated enzyme in the presence of various metal salts is ++ given in Table III. Mg was the most effective activator, 10 TABLE I Purification of D-Glucokinase Total Specific Fraction Activity Recovery Activity units/mg Efllfiéfi g protein Cell extract 401,000 100 2.2 Bentonite supernatant 229,000 57 15.1 Ammonium sulfate I 146,000 37 46.7 pH 4.4 supernatant 158,000 39 69.2 Ammonium sulfate 11 73,400 18 164 Sephadex G-100 49,100 12 1,230 DEAE-cellulose, fraction 24 7,000 ‘\ 4,000 n " " 25 7,000 4,360 " " " 26 4,420 6.6 3,360 n n H 27 4,310 4,100 n n . 28 3,580 3.500 *uMoles of D-glucose phosphorylated per hour. 11 Fig. 1. pH Optima of D—glucokinase. The routine assay was used except that the buffer composition and pH were varied as indicated, with the D-olucokinase (DPAF—cellulose frac— tion) concentration constant. The pH measurements were made on duplicate reaction mixtures with a Sargent DP pH meter €0u1pped with a Jenaer combination microelectrode. The pH did not change during the 5-minute assay period. Figure 1. pH OPTIMA OF D—GLUCOKINASE A. aerogenes t I00 5 1 ,A/ 1 .. Has 1 i 9 >_ 80 3‘ f \ varied [:- 0 g\ | > : frac- }: 9 8 1 ‘xare 2% [/8 1 ‘ so . 1 - | —— — '2 pa “2‘ fig . GLYCINE ) 1'— 1 098- j GLYCYLGLYCINE 1 1 0d 3? 4C) f i l 1 1 1 i 20 f 1 1 1 O 1 6 8 9 IO 12 13 TABLE I I Phosphoryl donor specificity of D-glucokinase The reaction mixture contained in a volume of 0.15 ml: 5 umoles of Dtglucose, 1 umole of MgC12, 0.1 umole of NADD, 0.5 umole of phosphoryl compound, 8 umoles of ciycyiolycine buffer (pH 7.5), purified D-glucokinase, and excess glucose 6-phosphate dehydrogenase. : Relative Phosphoryl donor phosphorylation rate” ATP 100 ITP 13 CTP 3 UTP 3 CT]? 0 ADP 0 acetyl phosphate 0 carbamyl phosphate 0 creatine phosphate 0 14 Fig. 2. Lineweaver—Rurk plot relating D-glucokinase reaction velocity to ATP concentration. The routine assay was used except that the ATP concentration was varied as indicated, with the D—glucokinase (DPAF- cellulose fraction) concentration constant. The MgC12 concentration was maintained at twice the ATP concen— tration. ASE: n:.< _ O.N m._ 0.. 0.0 O 0.01 O..- 5 l 1-111111,11I1:11.1 11;,1 1 .111 m“.— i i i i. _ . 34 mo“. _Ex 1 i Flgure 2 2 ¢ I 9 X m -.—-vI—\,YfA 1* m um. - m-m‘; .7 -e‘ l6 TABLE III Metal ion specificity of D-glucokinase The reaction mixture contained in a volume of 5 umoles of D-glucose, 0.1 pmole of NADP, 0.5 umole of ATP 8 umoles of glycylglycine buffer (pH 7.5), 1 umole of the metal salt, purified D-glucokinase (treated with 0.01 M EDTA, pH 7.0, and dialyzed by passage through Sephadex), and excess glucose 6-phosphate dehydrogenase. was initiated by the addition of D—glucokinase. 0.15 m1° The reaction Metal salt Relative phosphorylation rate MgSO4 NnClz CoCl2 NiSO 4 CaC12 S. Zn 04 None 100 100 43 24 1 1 I ‘- -—-.‘ v , 17 with Mn++ and Co++ being partially effective. Substrate Specificity- The glucose 6—phosphate dehydrogenase-linked assay was used to obtain an indication of speCifiCity by measuring the inhibition of phosphoryla- tion of 1 mM D-glucose in the presence of 100 mM concen- trations of other sugars. Inhibition was detected only with D—glucosamine and D-xylose. Compounds which caused if“ no inhibition were: 2-deoxy-D—g1ucose, o—methyl—D—giico- n side, L-glucose, D- and L—mannose, D-allose, D—altrose, D— and L~ga1actose, D- and L-fucose, L-rhamnose, D—gluconic acid, D-glucuronic aCid, D-galacturonic acid, D—sorbitol, D-mannitol, D— and L-arabitol, ribitol, xylitol, L-sorbose, sucrose, L-xylose, D—lyxose, D—ribose, D- and L- arabinose, D— and L— ribulose, and D— and L-xylulose, The observed inhibition With D—gluccsamine and D—xylose was competetive with Deglucose 1Fig5. 3 and 41, with the K31 being 0.4 mM for D-glucosamine (Fig.5; and 3 mM for D-xylose (Fig. 61. The nonspeCiiic pyruvic kinase—lactic dehydrogenase— linked assay .8; was used to measure possible phosphoryla- tion. Fig- 7 shows that D-fructose, D-mannose, and Z-deoxy— D—glucose were not phosphorylated in an assay which was suffiCiently sensitive to detect phosphorylation at 0.2% of the rate of phosphorylation of D—glucose. D—Glucosamine was phosphorylated at about 26% of the rate on D-glucose 18 Fig. 3. Lineweaver-Burk plot shOWing the relationship of D-glucose concentration to D—glucokinase reaction velocity in the presence of various concentrations of D-glucosamine. The routine assay was used except that the D-glucose and D~g1ucosamine concentrations were varied as indicated, with the D—glucokinase (DFAF-cellulose fraction) concen— tration constant. tip of eloCity osamine e and ed, IDCEH‘ F igure 3 . T l 7 o- GLUCOSAMINE‘ ° INHIBITION IO 6 '_ 1 V 1 1 '4 I 2 -IO 0 D— GLUCOSE (mM) 19 20 Fig. 4. Lineweaver-Burk plot showing the relationship of D-glucose concentration to D-glucokinase reaction velocity in the presence of various concentrations of D—xylose. The routine assay was used except that the D—glucose and D- xylose concentrations were varied as indicated, with the D-giucokinase (DFAF-cellulose fraction) concentration constant. F igure 4 . 6.7 mM 0 - XYLOSE 20 30 40 I D — GLUCOSE (mM) 21 22 Pig. 5. Kinetic plot for obtaining the Ki for D-glucosamine. The data are from the experiment described in Fig. 3. Figure 5 . I 1 o Ki (o-GLUCOSAMINE) = 4 x 10-4 M 50 pM o-GLUCOSE 8 _ /,,__ O 6 ‘M ‘3 /".7 "I’d" I I :71 o ,/|QO pM D-GLUCOSE / ""7"" M ' "IN—*‘fi .’ o 1 / 200 pM D—GLUCOSE O 0.5 |.O 1.5 D-GLUCOSAMINE CONCENTRATION (mM) 23 - ,vq .~v-‘— .._x_i_ W. ‘. _ O 5"“; "V—v..-“~ 24 Fig. 6. Kinetic plot for obtaining the K1 for D-xylose. The data are from the experiment described in Fig. 4. F igure 6 . u... __ A)“ 0/ ._.. ..*/. / 201‘0’ AMI: ; 1 1 . O I E 1 79'2””??? 1/ /. 1 | 1.4 -G 1 1 I 1 LU’COSE' I / -2 O 2 4 D -XYLOSE CONCENTRATION (mM) 25 6 26 Fig. 7. Specificity of D-glucokinase. Each cuvette contained in a volume of 0.15 ml: 10 umoles of glycylgl- cine buffer (pH 7.5), 0.5 umole of ATP, 1.0 umole of MgClZ, 0.5 umole of phosphoenolpyruvate, 0.05 umole of NADH, 0.15 umole of sugar, excess crystalline lactate dehydrogenase (containing pryuvate kinase), and purified D—glucokinase. The cuvette compartment was thermostated at 25°. The reaction rate was proportional with D— glucokinase concentration. Controls without ATP were negative. F igure 7 . I I SPECIFICITY OF GLUCOKINASE H20 H20 0- FRUCTOSE D—MANNOSE cylgl- 2.0 ADD MINUTES 27 21 g 1.81 o-GLucosg D—GLUCOSE 1'3 1 1 5 D-GLUCOSAMINE ' g D-GLUCOSE 1 0° 1.6 _ ' tr we —o GLUCOSE O ‘” 1 CD 4 1 t l 1.4 7' L \ 1 \\ 1 I \ 12 1 0 IO 20 0 IO 20 30 28 The addition of D-glucose to the negative cuvettes after 15 to 30 minutes resulted in a rapid decrease in absor- bance, verifying that D-glucokinase and the coupling enzymes were not inactivated or inhibited by the other sugars. Similar experiments With higher levels of D- glucokinase for increased senSitivity indicated that the following compounds were also not phosphorylated: D— and L-xylose, a-methyl—D-gluCOSide, L—glucose, L—mannose, L—fructose, D—allose, D—altrose, D— and L—galactose, D— and L—fucose, L-rhamnose, D-gluconic aCid, D-glucuronic acid, D-galacturonic acid, D-sorbitol, D—mannitol, D- and L—arabitol, ribitol, xylitol, L-sorbose, D—lyxose, D-ribose, D— and L-arabinose, D— and L-ribulose, and D— and L—Xylu— lose. From the data depicted in Fig. 8, the Km for D— glucose was determined to be 80 0M. Product inhibition- With the pyrUVic kinase-lactic dehydrogenase-linked assay (see Fig. 7), no inhibition of the phosphorylation of 1 mM D—glucose was observed in the presence of 10 mM D—glucose 6—phosphate, D—Mannose 6-ph03phate, although not a product, was also tested and found to give no inhibition at these concentrations. ADP inhibition was competitive With ATP (Fig. 91, With the K1 being 0.4 mM (Fig. 10). 29 Fig. 8. Lineweaver-Rurk plot relating D—glucokinase reaction velocity to D-glucose concentration. The routine assay was used except that the D-glucose concentration was varied as indicated, with the D—glucokinase (DPAF- cellulose fraction) concentration constant. 22c: mmoonqo .o O_.. 30 Figure 8. .2 m-o_ x m . $830-0 mo... Ex _ _ _ 3 v routin i 3 31 Fig. 9. Lineweaver-Burk plot showing the relationship of ATP concentration to D—glucokinase reaction veloCity in the presence of various concentrations of ADP. The routine assay was used except that the ATP and ADP concentrations were varied as indicated with the D—gluco— kinase (DEAF—cellulose fraction) concentration constant. The MgClZ concentration was maintained at twice the concentration of ATP plus ADP. Figure 9 . 1 I ADP INHIBITION 1 120 1 3.3 mM ‘ADP I _ I I 1 I l 1 I 1.3 mM ADP I __ ./i 0".“ Viv—J. NO ADP /" ‘ ./. 0.67 mM YADP V l 1 -I.O O | 0.5 ATP (mM) 32 IO |.5 33 Fig. 10. Kinetic plot for obtaining the K1 for ADP. The data are from the experiment described in Fig. 9. Figure 10 . I Ki (ADP) = 4 x 10'4 M / ,_.____ 20 __-_ _. .. i. —-——— I5 —_._._. .._ v .. '_ V. DD. The | _. __ '0 » ———— s -:/-;; ,,/ K. // /»// 6.7 mM ATP - '\A ’0’./ -2 o 2 4 6 8 A DP CONCENTRATION (mM) 34 35 Stability- Purified D-glucokinase was kept at room temperature for several days or at 0° (unfrozen) for several weeks without a significant loss of activity. It was unstable to storage in the frozen state at —20°. Product Identification The product of the D—glucokinase—catalyzed reaction was prepared by incubating in a microcuvette: 2.5 units of D-glucokinase (DEAE-cellulose fraction), 0.030 umole of D—glucose, 0.5 umole of ATP, 1.0 umole of MgClz, 0.2 umole of NADP, and 10.0 umoles of glycylglycine buffer (pH 7.5), in a volume of 0.15 ml. After incubation at 25° for 25 minutes, excess glucose 6-phosphate dehydrogenase was added. This resulted in an increase in abacrbance at 340 mu equivalent to the oxidation of 0.029 umole of D—glucose 6-phosphate. The further addition of excess phosphoglu- comutase did not result in a change in absorbance after correcting for dilution. Other experiments indicated that D-glucokinase and glucose 6—phosphate dehydrogenase were free from phOSphoglucomutase and 6-phosphog1uconate dehydro— genase. Thus, it was established that the product of the D-glucokinase—catalyzed reaction is D-glucose 6—phosphate and not D-glucose l—phosphate. Effect of Growth Substrate on D—Glucokinase Level The specific activity of D—glucokinase in extracts of cells grown on D—glucose—free (<0.0001%) nutrient broth 36 (0.5% Difco peptone, 0.3% Difco beef extract, pH 7.0) or on the mineral medium with 0.5% glycerol in place of D-glucose was the same as in extracts of cells grown on the D-glucose—mineral medium. Therefore, D—glucokinase may be considered to be constitutive in this organism. DISCUSSION Although several other kinases presumably stereo- specific for D-glucose have been reported (18-24), their existence and speCifiCity had not been established after extensive purification. After this work was completed, however, a report by Saito (25) described a D-glucokinase purified 113—fold from Brevibacterium fuscum. In addition, a kinase which phosphorylates D-glucose and D—mannose but not most other sugars has recently been purified ZOO—fold from rabbit liver (26). Specificity studies on the A. aerogenes D—glucokinase indicate that for a compound to bind at the D-glucose Site, it must possess an aldghydg group at carbon atom 1 and the D—glugg configuration at carbon atoms 2,3, and 4. The —OH at carbon atom 2 may be replaced by -NH2 (as in D-glucosa- mine) but not by —H (as in 2—deoxy-D—g1ucose). D-Xylose satisfies these criteria and competitively inhibits the phosphorylation of D—glucose, but is not itself phosphory— lated. To be phosphorylated, the compound binding at the 37 D'glucose site must also contain a hydroxmethyl group attached to carbon atom 5. Thus, the only compounds that have been demonstrated to be phosphorylated by this D-gluco— kinase are D—glucose and D-glucosamine. D-Glucokinase is constitutive in A. aerogenes PRL—R3 and presumably functions in initiating the metabolism of D-glucose and D-glucosamine. Hexokinases (ATP:hexose phosphotransferases) for other common hexoses such as D-mannose and D-fructose, however, have escaped detection in extracts of A. aerogenes PRL-R3, even though these com— pounds can be metabolized constitutively by this organism. A similar situation presumably eXists in Escherichia 921$. Fraenkel, Falcoz—Kelly, and Horecker (27) have described a mutant (FR—1) of E. ggli which lacks the specific D—gluco- kinase, and another mutant (MM—6) which, unlike the Wild type or mutant FR—l, is unable to grow on D-fructose. The genetic defect in mutant MM—6 has been postulated to be due to either (i) altered permeability (28), or (ii) lack of a "nonspecific hexokinase...which, for some reason, is diffi- cult to measure in extracts" (27). Since several enzymes have now been described which phosphorylate hexoses with phosphoryl donors other than ATP (6,7, 29—31), it is pOSSi- ble that not one, but several, nonspecific phosphotrans— ferases act in concert to phosphorylate hexoses in any one 38 organism. Because some of these enzymes-can use D-glucose phosphate as the phosphoryl donor (6, 30, 31), it is attractive to speculate that the stereospecific D-gluco- kinase described here functions not only in the initia— tion of the metabolism of D—glucose, but also indirectly in the metabolism of other hexoses for which ATP:hexose phosphotransferase activity has not been demonstrated. SUMMARY OF PART I A constitutive, stereospecific D-glucokinase was purified over 1,000—fold from extracts of A. aerogenes PRL-R3. Only D-glucose (Km= 8 x lO—SM) and D-glucosa- mine (Kin 4 x 10_4M) were phosphorylated. The enzyme was inhibited by D-xylose (competitive with D-glucose, Ki=3 x 10—3M) but not by 34 other sugars and related compounds tested. It was inhibited by ADP (competitive with ATP, Ki: 4 x 10_4M) but not by D—glucose 6-phosphate or D-mannose 6—phosphate. The pH optimum was 7.5 in glycylglycine buffer and about 8.9 in glycine buffer. Other properties studied were phosphoryl donor specificity, metal ion specificity, and stability. The product of D-glucose phosphorylation was identified as D-glucose 6-phosphate. PART II A Cyclic Pathway for the Metabolism of D—Mannose Because of the widespread occurrence of hexokinase and D-mannose 6-phosphate isomerase in yeast, animal tissues, and bacteria, it is generally believed that D—mannose is metabolized by phosohorylation with ATP to yield D-mannose 6—phosphate, followed by isomeriza— tion to D-fructose 6-phosphate. The constitutive hexo— kinase of A. aerogenes PRL—P3, however, has been purified over 1,000-fold and shown to be highly stereo— specific for D-glucose (see Part I). Attempts to demonstrate unequivocally the eXistence of D—mannokinase in this organism have conSistently yielded negative results in spite of the fact that D—mannose is metaboliz- ed constitutively. Rather, an apparent 2-epimerization of D—mannose to D-glucose was detected in extracts. in an effort to reconcile these observations, we have identified a seguence oF reactions which lead us to propose a cyclic pathway for the metabolism of D-mannose which is independent of the involvement of D—mannokinase. In addition to D-glucokinase, D—mannose 6—phosphate isomerase, and D—glucose 6—phosphate isomerase, the 39 40 pathway involves the participation of acyl phosphate:hexose phosphotransferase (5), which has now been purified several hundred fold and shown also to possess hexose phosphate: hexose phosphotransferase activity (7) (see Part III). This section of the thesis outlines the pathway and describes the experiments which led to its proposal. EXPERIMFNTAL PROCEDURE Growth of Organism— A. aerogenes PRL-R3 was grown aerobically at 303 for 18 hours and harvested by centrifu- gation. Unless otherWise specified, the D-glucose—mineral medium described in Part I was used. The peptone—beef extract medium used in one experiment conSisted of 0.5% Difco peptone and 0.3% Difco beef extract, pH 7.0. Preparation of Cell Extracts- Cell extracts were prepared by treatment of cell suspensions for 5 to 10 minutes in a Raytheon Model DF—lOl, 250 watt, 10—kc sonic OSCillator Circulated With ice water. The supernatant fluid, after removal of the cellular debris by centrifu— gation at 31,000 x g, was the crude extract. ReagenEi— Intestinal alkaline phosphatase and glucose oxidase (Glucostat) were obtained from the Worthington Biochemical Corporation. Clucose 6—phosphate dehydro— genase of suitable purity for the enzyme—coupled assays was obtained from a variety of commercial sources. 41 D”G141cokinase was the preparation described in Section I, and purified acyl phosphate:hexose phosphotransferase was the preparation described in Section III. D—Mannose (C.P. grade) was twice recrystallized (14) to remove interfering amounts of D-glucose. All other chemicals were used as obtained from commercial sources. Analytical Procedures- Spectrophotometric measure— ments of reduced pyridine nucleotides were made at 340 mp with a Gilford absorbance—recording spectrophotometer thermostated at 25°, using microcuvettes with a 1—cm light path. Manometric measurements were made using conventional techniques (32). Descending paper chroma- tography of sugars employed Whatman No. 1 paper (washed with 1N HCl and water) with 80% phenol as the solvent. The sugars on the chromatograms were visualized with silver nitrate (33) or with N,N—dimethy—pfphenylenediamine monohydrochloride (34). Radioactivity scans of paper chromatograms were made with a Nuclear—Chicago Model 1036 4-pi Actigraph II scanner, Nuclear-Chicago Model 162OCS analytical count ratemeter, and Sargent Model SRL recorder. D-Glucose was measured with glucose oxidase or by measuring NADP reduction in the presence of excess purified stereospecific D-glucokinase, glucose 6—phosphate dehydrogenase, and ATP. Other procedures were as described in Section I. 42 Enzyme Assays- D-Glucokinase and acvl phosphate:hexose phosphotransferase were assayed as described in Parts I and III, respectively. D—Clucose 6—phosphate isomerase was assayed spectrophotometrically by measuring NADP reduction in the presence of D-fructose 6—phosphate (containing limited D—glucose 6—phosphate), and glucose 6-phosphate dehydrogenase. D—Mannose 6-phosphate isomerase was assayed spectrophometrically by measuring NADP reduction in the presence of D—mannose 6-phosphate, D—glucose 6-phosphate isomerase, and glucose 6—phosphate dehydrogenase. RESULTS AND DISCUSSION Whole Cell Fermentation— Cells of A: aerogenes PRL—R3 grown on either the D—glucose—mineral medium or on a peptone-beef medium fermented D—glucose and D-mannose at equivalent rates (Fig. 11), indicating that the metabolism of these two hexoses is constitutive in this organism. Absence of D—Mannokinase— Several different assay procedures were used in attempts to detect the possible phosphorylation of D—mannose with ATP. These included measurement of the disappearance of reducing sugars (35) after removal of the phosphate esters with barium (36); measurement of acid production manometrically (37), spectrophotometrically (38), and by titration with NaOH to maintain a constant pH; and the measurement of ADP 43 Fig. 11. Fermentation of D-glucose, D—mannose, and D— fructose by cells of A. aerogenes PPL-R3 grown on D-glucose—mineral salts and on peptone-beef extract. Fach Warburg vessel contained in a volume of 0.5 ml: l0 tmoles of NaHCO3, 10 umoles of hexose, and washed cells (1.86 mg dry weight). The gas phase was 5% C02 in nitrogen, and the temperature was 30°. An endogeneous rate of about 6 ul of CO2 per hr per mg dry weight has been subtracted from the rates shown. Figure 11. mmzbzi m: o_ m o m. o_ m o mmoeosEa IX. 0. #053151 N _. o - o x..\ _ a _ . ON _ wmgzzaz -o 1W A1 mmwoaqono _ 2>>Om0 IwZOHdma 30.2242-.. 1 i i All mmOUDJoI o i 0... AP \m _- x i ._. 226% I mmooads _ i i L 0.? 1.1 1 1|1IHII 1 111fl1l11 _ _ _ ZO_._.<._.zm_>Em“_ limo ijI>> “ 9 O N 0 r0 0 v- iH913M A80 9W/303 1d 44 45 formation in a pyruvate kinase-lactate dehydrogenase—linked assay (13). Although the results obtained with the differ- ent methods varied, an apparent activity could always be detected in crude extracts by manometric and titrimetric assays. Attempts to purify the apparent D-mannokinase by various fractionation procedures, however, invariably led to a loss of activity. Typical data are shown in Fig. 12. The titrimetric assay for kinases showed activity on both D-glucose and D-mannose in the crude extract, but only on D—glucose in the ammonium sulfate fraction. Other fractions were also devoid of D—mannokinase activity. The use of EDTA, reduced glutathione, or mercaptoethanol during frac— tionation and assay had no effect on preserving D-mannokinase actiVity. D-Mannokinase activity was also not detected in particulate fractions of broken—cell suspensions. Assays with CTP, CTP, ITP, and UTP in place of ATP were also negative. Cell extracts prepared by procedures other than sonic Vibration, such as with a French pressure cell, also contain- ed no detectable D-mannokinase activity after fractionation. Although it is possible that a very labile or otherwise peculiar D-mannokinase does exist in A. aerogenes PRL-R3, the apparent activity that was detected in crude extracts can also be explained on a basis other than the direct phosphorylation of D-mannose with ATP. In the titrimetric . ' 'u‘L-“Ie-Wp' a» ‘ ———.- 46 Fig. 12. Titrimetric assay for D-glucokinase and D- mannokinase in a crude extract and an ammonium sulfate fraCtion. Fach reaction mixture contained in a volume of 1.64 ml: 40 pmoles of ATP, 80 umoles of MgClZ, 80 smoles of the indicated hexose, and extract (25 mg of protein for the crude extract and 20 mg of protein for the ammonium sulfate fraction). The pH was maintained between 7.2 and 7.4 with the periodic addition of Y"aOH. The temperature was 25°. Og. Figure 12 . mm._.32__2 . \. a\ $830-0 1M \ \. m 22an sommlsizi $8-9. 9 ON mm Om ON 0. 0 CV om ON 0_ msozmooozm . mmozzqz- o //..,.o\ .\o o o o _ oflb\ \ o\\ \ $83.90 . \o m . . \ .. a 84 a msozmooozm I o a ”K , _ \\\\\ . O \ \O . . D o\. \. Immozzazs . «\\ . . \\w .\ “$830-0 I . . . o . D .\ ko "Cw-GLUCOSE ”H. (can: EXTRACT) ! 0. TIME CONTROL :2 400 - 2 a \ I? 3 f 200 r- 4039" _ L .A+ m I I I I 5 no cu IS 20 400 - 3 - noun mcuamon g s 200 ~ km GLUCOSE m H—N—d M l _ L l 55 56 to D-glucose was indicated. inactivation of the D-Mannose to D-Clucose Conversion by Charcoal— Treatment of a crude extract With 10% and 20% charcoal (Darco G-60) caused a loss of the ability to convert D-mannose to D-glucose 53% and 100%, respectively, suggesting the involvement of a cofactor. This was not unexpected since other epimerases are known which require charcoal-adsorbable cofactors. For example, N-acyl-D- glucosamine 2-epimerase requires ATP (41), and UDP-galactose 4-epimerase requires NAD (42)= However, attempts to re- activate the charcoal-inactivated extract Wlth ATP, ITP, UTP, CTP, CTP, TTP, or NAD were unsuccessful. Furthermore, attempts to elute from the charcoal a cofactor which would reactivate the extract were unsuccessful. The pOSSibility existed that a protein was adsorbed by the charcoal, although analySis of the charcoal-treated extract for enzymes such as D-glucokinase, D—glucose 6-phosphate isomerase, and D— mannose 6-phosphate isomerase indicated that they were not adsorbed. The pOSSibility also eXisted that the suspected Z-epimerase acted on nucleOSide diphosphate derivatives of D—mannose and D-glucose rather than on the free sugars (43), and that the necessary sugar nucleotides were adsorbed on the charcoal. This scheme would require another enzyme, a transferase which would catalyze a glycosyl exchange of 57 D—mannose Wlth a nucleoside diphosphate—D—glucose to yield a nucleOSide diphosphate-D-mannose and D-glucose. Experi- ments which are described below, however, indicate that the converSion of D-mannose to D-glucose can be explained on a basis other than one involving two hypothetical enzymes. Inhibition of the D-Mannose to D-Clucose Conversion by Alkaline Phosphatase- When a crude extract was supple- mented With alkaline phosphatase and incubated with D- mannose, no D-glucose was formed (Table IV). This suggest— ed the partiCipation of phosphomonoesters in the D-mannose to D-glucose converSion. ATP—Dependence of the D—Mannose to D-Clucose ConverSion— Molecular sieving of a crude extract by passage through Sephadex C-ZS abolished its ability to convert D—mannose to D-glucose. The activity could be restored, however, by the addition of ATP to the reaction mixture (Fig. 16). The rate was maXimal at an ATP concentration of about 0 5 mM. At higher ATP concentrations, the D-glucose formed was phosphorylated to D—glucose 6-phosphate. Preliminary experiments on the stoichiometry of the reaction indicated that more than two moles of D-glucose were formed per mole Of ATP, suggesting that the ATP was acting catalytically. Thus, the reaction had a superfiCial resemblance to the ATP-dependent 2—epimerization of N-acyl—D-glucosamine Effect of added alkalineyphosphatase on 58 TABLE IV the formation of D-glucose from D— mannose by a crude cell extract The complete reaction mixture contained in a volume of 1.0 ml: 60 umoles of glycylglycine buffer (pH 7.5), 50 umoles of D—mannose, extract (11.u mg of protein), and 2 mg of alkaline phos— l2 umoles of MgCl2, crude cell phatase. Controls were minus phosphatase or D-mannose as indicated. After incubation for the times indicated, the reaction mixtures were heated in a boiling water bath for 2 minutes, cooled, and centrifuged. of the supernatant solutions were then assayed for D-glu- cose with D-glucokinase-glucose 6-phosphate dehydrogenase. Aliquots c 03 D-Glucose Formed +>o .253 Minus Phosphatase Plus Phosphatase 36% g + D—Mannose — D-Mannose + D—Mannose — D-Mannose H mumoles/mg mpmoles/mg mumoles/mg mumoles/mg Minutes protein protein protein protein 0 0 0 7 7 20 56 0 17 20 40 86 O 23 20 60 126 0 23 23 "« 31’"'-'.."" -, g"":'£-.‘-".'ra_ w... w v..— _ 59 Fig. 16. Inactivation of the ability of a crude extract to convert D-mannose to D-glucose by chromatography on Sephadex G-25 and reactivation with ATP. Crude extract (10 ml) was passed through a column (20 x 3.5 cm) of Sephadex G-25. The activity of the resulting protein fraction was then tested in reaction mixtures consist- ing of 60 umoles of glycylglycine buffer (pH 7.5), 12 umoles of MgClz, 50 umoles of D-mannose, crude extract (2.4 mg of protein), and ATP in amounts varying from O to 5 umoles, in a volume of 1.0 ml. After incubation at 25° for 60 minutes, the tubes were heated in a boiling water bath for 2 minutes, cooled, and centrifuged. Aliquots of the supernatant solutions were then assayed for Dhglucose and D-glucose 6-ph05phate by the use of Deglucokinase and glucose 6-phosphate dehydrogenase. Ge6-P, D-glucoSe 6ephosphate. oiling sayed of Figure 16. pMOLES FORMED/ML/HR 0.5 0.4 0.3 SEPHADEX G-25 - TREATED EXTRACT GIIUCOSE + G-6—P ! I l .ar'1'—_':'_—““-—..= GLUCOSE l I l T }____ l 1 T I O ATP CONCENTRAHON hnM) r / /'/j:° _ V ,/'/ i 61 recently described by Ghosh and Roseman (41). However, experiments which are described below indicate that the D-mannose to D—glucose converSion observed in extracts of A. aerogenes PRL—R3 was mediated by the concerted action of several enzymes rather than a single ATP-dependent enzyme. Evidence that D—Clucose was not Derived from D-Glucose Phosphate by the Hydrolytic Action of a Phosphatase— In View of the ATP-dependence and alkaline phosphatase—senSitiVity of the D-mannose to D-glucose conversion, the pOSSibility existed that D—mannose was somehow phosphorylated to D—mannose 6-phosphate and converted to D—glucose 6—phosphate and D-glucose l—phosphate by isomerase- and mutase-catalyzed reactions. The D—glucose might then arise from the hydrolySis of D—glucose 6-phosphate or D-glucose l—phosphate. FVidence which militates against this is illustrated in Fig. 17; a crude extract formed D-glucose from D-mannose but not from the phosphate esters of D-mannose, D—glucose, or D—fructose. Measurement of the Conversion of D-Mannose to D-Clucose 6—Phosphate in a Continuous Spectrophotometric Assay; Dicov— ery of Acyl PhOSphate:Hexose Phosphotransferase— Pecause crude extracts contained an active D—glucokinase with a high affinity for D-glucose (Km: 8 x 10'5M) , the ATP—dependent conversion of D—mannose to D-glucose could conveniently be 62 Fig. 17. Measurement of the D—glucose formed from D-mannose and from hexose phOSphates in a crude extract. The reaction mixtures contained in a volume of 7 ml: 100 “moles of hexose phosphate or 500 umoles of D-mannose, and crude extract (252 mg of protein), adjusted to pH 7.5. One-ml samples were removed at time intervals, heated in a beiling water bath for 2 minutes, cooled, and centrifuged. Aliquots of the supernatant solutions were then assayed for D—glucose Wlth glucose OXidase. M—6—P, D-mannose 6-ph05phate; C-l—P D-glucose l-phosphate; F—6—P, D—fructose 6-phosphate; G—6uP, D—glucose 6-phosphate. mMHDZE ON_ Om Om Om . 0 l4; ca: _ .4 . Au 5 mood WfiHNWVMAL .0 23 a o-m In 2 _ mu nu nu no Co as w, 4H mu no u. a: co / mu no .a no 0 l mm mm a mu \\ mum _\\\\ i w\ ms mmozzIaemo< a -o .. wmooao- a $8224: A; x 3:»? YO. Mk 3. g IOO — — 20 5 a: < CL so e — I0 I O “443}1 \:\\lo “AAA-AAAAM 0 FRACTION NUMBER 91 92 ‘TABLE VII Purification of acyl'phosphgte:hexoSefphosphptrapsferase Fraction Total Yield Specific Activity Activity HEEEE A Units/mg Cell extract 1,570b (76) 0.11 Protamine sulfate 2,060b' 100 0.29 Ammonium sulfate I 1,24010 60 0.50 Heat 1,250 61 1.1 Ammonium sulfate II 1,300 63 2.5 Sephadex C—200 294 14 6.5 DEAE-cellulose 174 8.5 78 a pMOles Of D-glucose phosphorylated per hour; corrected for the portion Of fractions not used for further purifica- tion. b Corrected for the 6~phosphogluconate dehydrogenase contribution by dividing the Observed rates by 2. 93 Fig. 23. pH Optimum Of acyl phosphate:hexose phosphotrans- ferase. The routine assay was used except that the buffer composition and pH were varied as indicated, with the enzyme concentration constant at 0.03 unit per cuvette. The pH measurements were made on duplicate reaction mixtures. the pH did not vary with time during the 5—minute assay period. ohotrane 1e buffer the vette. on mixtures assay RELATIVE ACTIVITY Figure 23. IOO 80 60 4O 20 pH - ACTIVITY PROFILE 94 .\ / c ,0 0/8. I I 8/° i ,o I / I /§ I 9 o GLYCINE o GLYCYLGLYCINE 0 7 8 9 pH 95 the presence Of various phosphoryl donors are given in Table VIII. The Km and Vmax values were determined for four Of the more active phosphoryl donors. The Km values for acetyl phosphate, carbamyl phosphate, and D—mannose 4 6-phosphate were essentially equal, at 4 x 10- M, whereas the Km value for D-ribose Sephosphate was 5-fold larger, . SIS! f 4" at 2 x 10‘3M (Fig. 24). The Vmax values were equal for acetyl phOSphate and carbamyl phosphate, whereas the values for D-mannose 6-phosphate and D-ribose 5—phosphate were “L: .931 about 75% and 31% (Fig. 24), respectively, of the Vmax value for acetyl phosphate and carbamyl phosphate. Phosphoryl Accgptor Specificity— Using spec1fic enzyme- coupled assays with acetyl phosphate as the phosphoryl donor, the phosphotransferase was demonstrated to phosphorylate D-glucose, D—mannose, and D-fructose at carbon atom 6, and D—mannitol at carbon atom 1. The apparent Km values were determined to be 1.6 x 1074M for D-glucose, 1.2 x 10'2M for D-mannose, about 0.3 M for D—fructose, and 6.7 x 10-2M for D-mannitol (Figs. 25 and 26). More recent experiments described below indicated that in the case Of D-fructose, D-fructose l—phosphate rather than D-fructose 6-phOSphate was the predominant product. The Vmax values for the phos— phorylation Of D—glucose, D-mannose, D-fructose and D-manni— tOl With acetyl phosphate, Within the limitations Of 96 TABLE VIII Phosphoryl donor specificity of phosphotransferase The standard assay (with D-glucose as the phosphoryl acceptor) was used except that the phosphoryl donor pmole) was varied. Phosphotransferase fraction) was 0.156 unit (1.8 pg of protein.) (DFAF-cellulose General Type Specific Example Relative phosphory— lation rate a Acyl phOSphate Fnol phosphate Hydroxyalkyl phosphate Alkyl triphosphate Alkyl perphosphate Phosphoramidate Inorganic pyrophosphate Acetyl phosphate Carbamyl phosphate Phosphoenolpyruvate D—Mannose 6-phosphate D-Fructose 6-phosphate D-Ribose 5-phOSphate D-Fructose l-phosphate a—D-Clucose l-phosphate D-Gluconate 6-phosphate D-Sorbitol 6—phosphate D-Mannitol 1-phosphate a-D-Mannose 1-phOSphate a—D-Calactose l—phosphate o-Clycerol phosphate D-Clycerate 3-phosphate ATP ADP Creatine phosphate Inorganic orthophosphate 100 100 0 71 25 18 15 OOOOONNU‘I U'IU‘I V‘WJ 97 Fig. 24. Phosphoryl donor kinetic constants for phosphotrans- ferase. The routine assay was used except that the phos— phoryl donor was varied as indicated, with the acyl phosphate: hexose phosphotransferase (DEAE-cellulose fraction) concen- tration constant at 0.092 unit. Figure 24. I I PHOSPHORYL DONOR KINETIC CONSTANTS I PHOSPHORYL DONOR (mM) 98 I I PHOSPHORYL Km REL. _20 l____ 4 DONOR (mM) Vmax 9 ACETYL-P 0.4 Ioo . I CARBAMYL-p 0.4 .00 7’ <——0I!- RIBOSE 5-P o-M-G-P 0.4 75 .-.5 _- ': L __ o-R-S-P 2.2 3| I I I I I I t ' V __o -NIANNOSE- -§;E AAAAAAA . ______ I I\ / /../ ”” zI. /9’ ’00”’J.I a'D”1{\ h ’6 *‘w‘ II-————-- _. ° ’\ I CARBAMYL- P (x) ACETYL- P(o) I I I I 2 3 .J 7.1.,1’! _.___ ' v‘..-—1‘,‘.—-— 99 Fig. 25. Lineweaver-Burk plot relating phosphotransferase reaction velocity to substrate concentration. The routine assays were used except that the substrate was varied as indicated with the phosphotransferase (DEAE-cellulose fraction) concentration constant at 0.108 unit per cuvette. Figure 25. WV.‘ ._‘.rivr --~.. . :tadoozv m _ OOQO. OOQm O Oms 00m Com 0 Om Om O_ O . I I - I o i m i m i m.O u onv , q.0 - on> _.C u Kore.) . ,2 q-o_xo..-&.._ no. 2 mo.xm._.5x \m .m zmottx . \mo K mmoonzéa “EOE/22.O \ _ $9.535 \om \ . w m 0 \ iv d O_ o Om \0 i _ o i \ _ 3 . \\\W .w m_ fink \O _ M o m M x. .m cm oo. .. 0 i0. 0 . . .mm o imm. mmqmmumz 113 114 Fig. 30. Kinetic plOt for obtaining the K1 for D-mannose using either D-mannose 6-phosphate or acetyl phosphate as the phosphoryl donor. The data are taken from the experi- ment described in Fig. 29. b.— Figure 30. T I I I Ki (D-MANNOSE) = 12 x IO‘2 M - I 40 O 30 ‘ l “25 pM o—GLUCOSE / D-MANNOSE-6 £046 -P . I;° '500 )JM D—GLUCOSE J “f—l/w’ - I ...—O -IO 0 IO 20 30 D—MANNOSEA CONCENTRATION (mM) 115 116 Fig. 31. Lineweaver—Burk plot showing the relationship of D-glucose concentration to reaction velocity at constant concentrations of acetyl phOSphate and D- mannose 6-phosphate. The data are taken from the experiment described in Fig. 29. 22:: mmooaqo I o _ N- 0- 0- ‘m o\\ meFm0< _ .P . \O . m_ I A - , - - . - - I mZxamoraémmozzqzs- . 0N - - I. - 2 0-0. x 0.. 0.. 300002242-.. 0 3 x 2 w 0. x 0.. 0.. ..-..Cmo.‘ m . A Q XUE u - $0030 . > 20200 .m. -. M25302 £202.10on F I . . mm gigg? gig}? .geggégg ago/”NA «23,3 / 13-8830 ” I I s I .. _. _ M g m .. ._ -...m _ . m I” _ ..... ' i _ _. I 8. ,7 i 4. w I. w r... m. .. oo~ SCEIS 125 'wk. - .- 126 TABLE IX Stoichiometry of the D-mannose 6—phosphate: D—glucose 6-phosphotransferase reaction, and D-glucose 6-phosphatase activity of phosphotransferase The reaction mixture in Experiment 1 contained in a volume of 4.0 ml: 320 pmoles of glycylglycine buffer (ph 7.5), 16 pmoles of D—glucose 6-phosphate, 800 pmoles of D-mannose, and 11.3 units of phosphotransferase (DEAE- cellulose fraction). Samples (0.5 ml) were taken at O, 15, and 30 minutes, centrifuged, and the supernatant used for analysis. The reaction mixture in Experiment 2 was minus D-mannose. Analysis of a control without enzyme indicated no changes from the O-time values. The incubation temperature was 25°C. (I) (D (l) (D-r-i U) U) U) (DD-I U) 0 O O O (l) O O C. C+ mp 5m 3 cm : E13 r—il r—l (Ul (1304 -HC mm w 20 2| Ei-H I I I I I ‘r-i IKO E Q Q o m D I Aumoles/ml Experiment 1 D—Glucose-6-P 15 —1.58 1.79 1.18 0.35 1.53 + D-mannose 30 —2.01 2.06 1.47 0.50 1.9? Experiment 2 15 —O.72 0.69 D-Glucose—6-P 30 -1.04 1.17 W 127 6-phosphotransferase in extracts of cells grown on D- glucose-free (<0.0001%) nutrient broth (0.5% Difco peptone, 0.3% Difco beef extract, pH 7.0) or on the mineral medium with glycerol in place of D—glucose was the same as in extracts of cells grown on the D—glucose-mineral medium. Therefore, it may be concluded that the enzyme is constitu— tive. Stability of Phosphotransferase— The most highly purified fractions of phosphotransferase have been stored at —20° for seven months with repeated thawing and freezing with no detectable loss of activity. , .29 ‘Pm. ”an“: mun-— Yew u. .4 III ll." ll’urlrlllllll III I I 128 DISCUSSION The reported data indicate that a single enzyme catalyzes all of the following reactions: (i) acyl phosphate + hexose+ organic acid f hexose phOSphate (ii) Hexose phosphate A + hexose Bt+hexose phosphate B+ hexose A (iii) hexose phosphate + H20» hexose + Pi (iv) acyl phosphate + H20 +organic acid + Pi For the phosphotransferase reactions, the phosphoryl donor may be either an acyl phosphate (acetyl phosphate or car- bamyl phosphate) or certain hydroxyalkylphosphates (e.g., D-mannose 6—phosphate or D-glucose 6eph05phate). Other hydroxyalkylphosphates (e.g., a-D—giucose l-phosphate, D-sorbitol 6-phosphate, or a-glycerOI phosphate), however, show little or no activity as phosphoryl donors. Other compounds (e.g., ATP, creatine phosphate, phosphoenolpyruvate and inorganic pyrophosphate) which have high phosphoryl transfer potential with certain enzymes also have no activity with this phosphotransferase. Although D—mannitol has been shown to serve as a phosphoryl acceptor, hexoses such as D-glucose and Demannose have higher affinities (lower Km values). Because of this fact, and because higher rates and higher affinities were observed with acyl phosphates and certain hexose phosphates than with other phosphoryl donors, the enzyme has 129 been named acyl phosphate (hexose phosphate):hexose phosphotransferase. The reversibility of reaction (ii) was demonstrated with D-mannose and D—glucose and their respective 6— phosphates. The irreverSibility of reaction (iii) was demonstrated by the inability of Pi to serve as a phosphoryl donor in the synthesis of D—glucose 6—phosphate. The irreverSibility of reactions (1) and (iv) was not determined experimentally, but would be predicted from the reported values for the free energies of hydrolySis of the phosphoryl compounds (51). The phosphotransferase-catalyzed hydrolysis of D— glucose 6—phosphate in the absence of an organic phosphoryl acceptor (D-mannose) was shown to be about half of the rate of D—glucose 6—phosphate disappearance in the presence of D-mannose. Since the hydrolysis of D—glucose 6-phosphate was inhibited about 50% in the presence of D—mannose, the hydrolase actiVity amounted to only about one-third of the phosphotransferase activity. Thus, the phosphotransfer— ase described here differs from "phosphatases" in three important respects: (1) the hexose phosphotransferase actiVities of phosphatases are generally less than the hydrolase activities (31, 52, 53); (ii) the Km values of phosphatases for organic phosphoryl acceptors are usually 130 large, often approaching l M (31, 54-58), whereas the Km of this phosphotransferase for D-glucose is low (4 x lO-4M); and (iii) the substrate specificities of phosphatases which can effect phosphotransferase reactions are usually broad (55, 57, 59, 60), whereas with this phosphotransferase it is relatively narrow. A possible exception to the last statement may be D-glucose 6-phosphatase, which possesses perphosphatezhexose phosphotransferase and hexose phosphate: hexose phosphotransferase activities (31). As has already been discussed in Part II, a physiologic- al role for the acyl phosphate (hexose phosphate):hexose phosphotransferase described here is readily apparent. A. aerogenes PRL-R3 can metabolize D—mannose constitutively yet apparently possesses no D-mannokinase (ATP:D-mannose 6-phosphotransferase). The described phosphotransferase, therefore, could substitute for D-mannokinase by phosphoryla- ting D-mannose with D—glucose 6-phosphate, acetyl phOSphate, or carbamyl phosphate. A. aerogenes PRL—P3, like E. coli (27), also seems to lack D-fructokinase. Whether the phosphotransferase described here can phosphorylate D-fructose to D—fructose 6-phosphate at a suitable rate to be metabolically significant is doubtful, because of the apparent high Km for D—fructose (about 0.3 M). However, more recent experiments revealed that D—fructose 131 l-phosphate rather than D—fructose 6-phosphate was the predominant product. It is possible that the Km for D— fructose is lower than 0.3 M when it binds in a pOSition suitable for phosphorylation at carbon atom l. Liss, Horwitz and Kaplan (61) have provided evidence that D-manitol is metabolized in A. aerogenes through D-mannitol l—phosphate. However, a kinase for D—mannitol could not be demonstrated (61). Consequently, the phosphotransferase described here becomes a candidate for initiating the metabolism for D—mannitol in this organism. Several other enzymes which phosphorylate hexoses with phosphoryl donors other than ATP have recently been described. These other phosphoryl donors include phosphoenol— pyruvate (29), D—glucose l—phosphate (30), phosphoramidate (62), inorganic perphOSphate (31), and various nucleOSide di- and triphosphates (31). It seems likely that in time still other phySiologically Significant phosphoryl donors (e.g., 1,3—diphosphoglycerate) for hexoses will be dis— covered. SUMMAPV 0]“ PART III A new enzyme, acyl phosphate (hexose phosphate):hexose phosphotransferase, was purified several hundred fold from extracts of A. aerogenes PPL—R3. 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