GLYCGSEéAfiEiegfi'LESFRATE WFfiACHGNgz GLYCC‘SEE’JE MQDEL HYEEQLY’Sfi RATES AND LYSQZYME-SUBSTRAYE EEACTEGNS Thesis Ecr- ‘ciw Degree of: p11. D. MICHEGAN STATE UNWEESITY Donald Rymbrandt 1967 Inu- n.‘_._ LIE}? b‘:a3? -. Laryegsity I'. 74"— This is to certifq that the thesis entitled Glycosidase-Substrate Interactions: Glycoside Model Hydrolysis Rates and Lysozyme-Substrate Reactions presented by Donald Rynbrandt has been accepted towards fulfillment of the requirements for Ph.D.l degree in Biochemistry lav/Am C lie; iv 17 Major profes’sor Date January 15; 1968 0-169. / 214, \ PF 6 .;’-‘fi ' 3"}?!- '-“' ~~N-di+ - 1'1 MICHIGAN STATE UNIVERSITY DEPARFIJEIIT CF BICCHSJISTRY EAST LANSING, MICHIGAN ABSTRACT GLYCOSIDASE-SUBSTRATE INTERACTIONS: GLXCOSIDE MODEL HXDBOLYSIS RATES AND LYSOZYME-SUBSTRATE REACTIONS by Donald Bynbrandt Several acetal, ketal, and 2-substituted tetrahydro- pyran glycoside model compounds were synthesized. These included 2-methylthio-, 2-methoxy- and 2-ethoxy-acetaldehyde diethyl acetal, u-methylthiobutraldehyde diethyl acetal, 5- methylthio- and 5-methoxy-2-pentanone diethyl ketal, and 2- ethoxy-, 2-(2-methylthioethoxy)-, 2-(2-methoxyethoxy)-, and 2-(2-pyridinyl-ethoxy)-tetrahydropyran. The hydronium ion-catalyzed hydrolysis rates of these glycoside model compounds were determined by spectrophoto- metric and hydrogen peroxide-oxidative methods. A comparison of these hydrolysis rates indicates that the sulfur atom of 2-methylthioacetaldehyde diethyl acetal increases the hydrolysis rate of that acetal by anchimeric assistance and sulfonium ion formation. Extrapolation of this mechanism to glycosidase catalysis indicates that a sulfonium ion-linked glycosylated enzyme intermediate is possible. These results were extended by an investigation of the reaction of hen's egg white lysozyme with soluble low- molecular weight substrates prepared from m, lysodeikticus cell wall. The peptide-containing lysozyme substrates formed a stable enzyme-substrate complex which could be purified and Donald Rynbrandt analyzed when the lysozyme-substrate reaction was quenched with guanidine hydrochloride, dithiothreitol, and N-ethyl maleimide. This complex may be one of the intermediates in lysozyme-substrate reactions. GLYCOSIDASE-SUBSTRATE INTERACTIONS: GLYCOSIDE MODEL HYDROLYSIS RATES AND LYSOZYME-SUBSTRATE REACTIONS By Donald Rynbrandt A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1967 é Uri: a 4 ‘3 5’; 0’6“ <3 ACKNOWLEDGMENT The author wishes to eXpress his appreciation to Dr. J. C. Speck, Jr.. for his assistance and guidance throughout the course of this study. The technical assistance of Miss Doris Bauer is also greatly appreciated. The support of a National Institutes of Health predoctoral fellowship is grate- fully acknowledged. ii To Kathy iii TABLE OF CONTENTS Page INTRODUCTION Acetal and Ketal Hydrolyses . . . . . . . . . . . . 1 Lysozyme Catalysis . . . . . . . . . . . . . . . . 2 EXPERIMENTAL Reagents . . . . . . . . . . . . . . . . . . . . . 13 Preparations Preparation of Preparation of Preparation of Diethyl Acetal Preparation of Acetal . . . . Preparation of Diethyl Acetal Preparation of Preparation.of Preparation of Preparation of Preparation of Ketal o o o 0 Preparation of Diethyl Ketal Preparation of Ketal o c o 0 Preparation of 2-Methylthioacetaldehyde . . 2-Hydroxyethyl Methyl Sulfide 2-Methylthioacetaldehyde 2-Methoxyacetaldehyde Diethyl 4-Methy1thiobutraldehyde 5-Hydroxyvaleraldehyde . . . valeraldehyde Diethyl Acetal ZéPentanone Diethyl Ketal . . 5-Chloro-2quntanone . . . . 5-Chloro-2-Pentanone Diethyl 5-Methylthio-2—Pentanone 5-Methoxy-2-Pentanone Diethyl Z-Ethoxytetrahydropyran . . . iv 17 18 19 20 20 2h 2# 24 25 25 26 27 27 Page Preparation of 2-(2-Methoxyethoxy)-Tetrahydro- Pyran o e o c o c c o o c o c o o o c e o o 0'. o 27 Preparation of 2-(2-Methylthioethoxy)- Tetrahydropyran o o o o o o o o o o c c o o o o o 28 Preparation of 2-(2-Pyridinylethoxy)- TetrahydrOpyran o c o o o o o o c o o o o o o o o 28 Preparation of Hydrolysis Buffers . . . . . . . . 29 Determination of Hydrolysis Rates . . . . . . . . 31 Determination of Acetal. Ketal, and Tetrahydro- pyran Hydr01y31s Rates 0 o o o o o o c e o o o c 33 Determination of Tetrahydropyran Ether Hydr01y818 Rates 0 o e c c e o o c o o o o o o 0 3“ Rate of Ethanol Formation in the Hydrolysis of 2-Methylthioacetaldehyde Diethyl Acetal . . . . . 36 Rate of Ethanol Formation in the Hydrolysis of 5-Methy1thio-2—Pentanone Diethyl Ketal e o o o o 37 Lysozyme Preparations Preparation of M, Mysodeikticus Cell Walls . . . 37 Hydrolysis of M, lysodeikticus Cell Walls . . . . 39 Automated Gel Filtration Apparatus . . . . . . . 41 Preparation of M, lysodeikticus Cell Wall Disaccharide, Tetrasacchar de, and "Fraction 1" Hexasaccharide-Glycopeptide Mixture . . . . . . . 4“ Preparation of M, lysodeikticus Cell Wall Hexasaccharide and Glycopepti e GP-l . . . . . . 50 Preparation of M, gysodeikticus Cell Wall Glycopeptide GP-Z o c o o c c o o o c c o o c o o 52 Purification of Nchetylglucosamine Tetramer . . 58 Purification of N-Acetylglucosamine Hexamer . . . 58 Preparation of N-Ethyl Maleimide-Inactivated Lysozyme O O O O O O O O O O O O O O O O O O O O 59 Page Preparation of Reduced, Carboxymethylated Lysozyme O O O O O O O O O O O I O O O 0 O O O O 60 Detection of Muramic Acid and Glucosamine in Lysozyme-Substrate Reaction Mixtures . . . . 61 Non-Aqueous Lysozyme-Substrate Reactions Using Dimethyl Sulfoxide Quenching . . . . . . . 62 Non-Aqueous Lysozyme-Substrate Reactions Utilizing Urea Quenching . . . . . . . . . . . . 63 Aqueous Lysozyme-Substrate Reactions . . . . . . 65 Effect of Hydroxylamine on the Lysozyme- Glycopeptide GP-Z Reaction . . . . . . . . . . . 68 RESULTS Acetal, Ketal, and Tetrahydropyran Ether Hydrolyses Hydrolysis Rate of 2-Methylthioacetaldehyde Diethyl Acetal in Dioxane-Water . . . . . . . . 7O Hydrolysis Rate of Z-Methoxyacetaldehyde Diethyl Acetal in Dioxane-Water . . . . . . . . 7i Hydrolysis Rate of 2-Ethoxyacetaldehyde Diethyl Acetal in Dioxane-Water . . . . . . . . 72 Hydrolysis Rate of h-Methylthiobutraldehyde Diethyl Acetal in Dioxane-Water . . . . . . . . 75 Hydrolysis Rate of Valeraldehyde Diethyl Acetal Acetal in Dioxane-Water . . . . . . . . . 76 Hydrolysis Rate of 5-Methoxy-2-Pentanone Diethyl Ketal in Dioxane-Water . . . . . . . . . 77 Hydrolysis Rate of 5-Methylthio-2-Pentanone Diethyl Ketal in Dioxane-Water o o c c o c o o o 80 Hydrolysis Rate of 2-Pentanone Diethyl Ketal in Dioxane-water . Q Q . Q . Q Q O O O Q . 8n Hydrolysis Rate of Z-Ethoxy-Tetrahydropyran inDioxane-Water.......o......o. 85 Hydrolysis Rate of 2-(2-Methylthioethoxy)- Tetrahydropyran in Dioxane-Water . . . . . . . . 86 vi Hydrolysis Rate Tetrahydropyran Hydrolysis Rate Tetrahydropyran Hydrolysis Rate Tetrahydropyran Ethanol Release Ethanol Release During the Hydrolysis of of in of in of in During the Hydrolysis of 2-Methylthioacetaldehyde Diethyl Acetal 2-(2-Methoxyethoxy)- Dioxane-Water . 2-(2-Pyr1d1ny1ethoxy)- Water . 2-(2-Methoxyethoxy)- Water a 5-Methylthio-2-Pentanone Diethyl Ketal . Lysozyme-Substrate Reactions Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reaction Mixtures which were Dimethyl Sulfoxide Quenched . Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reaction Mixtures. runs using reduced, N-ethyl maleimide-lysozyme Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reaction Mixtures. runs using reduced, carboxymethylated lysozyme Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reactions Utilizing Urea Quenching . ' Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reactions. utilizing glycerol . . . . Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reactions. without glycerol . Substrate Concentrations in Aqueous Lysozyme- Substrate Reactions Substrate Concentrations in Aqueous Lysozyme- Substrate Reactions. Effect of Hydroxylamine on the Lysozyme- Blank runs . Blank runs Blank runs Blank Blank Glycopeptide GP-2 Reaction . . . . . . . . . . DISCUSSION AND CONCLUSIONS . . . BIBLIOGRAPHY O O O O O O O O O O O O O O O O O O O 0 vii Page 87 88 89 91 95 96 97 98 99 100 101 102 103 104 105 122 LIST OF TABLES Table I Preparation of Acetate Buffers for Ketal Hydrolysis Rate Determinations . . . . . . . II Sephadex G-15 Gel Filtration of M, lysodeikticus Cell Wall Saccharides . . . III Amino Acid Analysis of Fraction 1 . . . . . IV Amino Acid Analysis of M, lysodeikticus Cell Wall Saccharide A-1 and Glycopeptides GP-landGP-Z00000000000000. VI Hydrolysis Rate of 2-Methylthioacetaldehyde Diethyl Acetal in Dioxane-Water . . . . . . VII Hydrolysis Rate of 2-Methoxyacetaldehyde Diethyl Acetal in Dioxane-Water . . . . . . VIII Hydrolysis Rate of Z-Ethoxyacetaldehyde Diethyl Acetal in Dioxane-Water . . . . . . IX Hydrolysis Rate of 2-Methy1thiobutraldehyde Diethyl Acetal in Dioxane-Water . . . . . . X Hydrolysis Rate of Valeraldehyde Diethyl Acetal in Dioxane-Water o o o e o o o o o o XI Hydrolysis Rate of S-Methoxy-Z-Pentanone Diethyl Ketal in Dioxane-Water . . . . . . . XII Hydrolysis Rate of 5-Methylthio-2-Pentanone Diethyl Ketal in Dioxane-Water . . . . . . . XIII Hydrolysis Rate of 2-Pentanone Diethyl Ketal in Dioxane-Water o o o o o o o c o o o XIV Hydrolysis Rate of 2-Ethoxy-Tetrahydropyran XV Hydrolysis Rate of 2-(2-Methylthioethoxy)- Tetrahydropyran in Dioxane-Water . . . . . . XVI Hydrolysis Rate of 2-(2-Methoxyethoxy)- Tetrahydropyran in Dioxane-Water . . . . . . viii Table XVII XVIII XIX XXII XXIII XXIV XXVI XXVII Hydrolysis Rate of 2-(2-Pyridinylethoxy)- Tetrahydropyran in Water . . . . . . . . . . Hydrolysis Rate of 2-(2-Methoxyethoxy)- Tetrahydropyran in Water 0 o o o o e o o c o Substrate Concentrations in Nonquueous Lysozyme-Substrate Reaction Mixtures which were Dimethyl Sulfoxide Quenched . . . . . . Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reaction Mixtures. runs using reduced, N-ethyl maleimide- treated lysozyme . . . . . . . . . . . . . . Blank Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reaction Mixtures. runs using reduced, carboxymethylated lysozyme suspended in glycerol . . . . . . . Blank Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reactions Utilizing UreaQuenching......o........ Substrate Concentrations 1n Non-Aqueous Lysozyme-Substrate Reactions. Blank runs utiliZIng glycerol 0 c o c o o c o o o o o o Substrate Concentrations in Non-Aqueous Lysozyme-Substrate Reactions. Blank runs WithOUtglycerOloooocoocoooooo Substrate Concentrations in Aqueous Lysozyme-Substrate Reactions . . . . . . . . Substrate Concentrations in Aqueous Lysozyme-Substrate Reactions. Blank runs . Effect of Hydroxylamine on the Lysozyme- Glycopeptide GP-Z Reaction . . . . . . . . . ix 96 97 98 99 100 101 102 103 104 Figgre 10 LIST OF FIGURES Automated Gel Filtration Apparatus . . . . . Automated Gel Filtration of M, lysodeikticus Saccharides . . . . . . . . Chromatography of M, lysodeikticus Glycopeptlde GP'2 0 e o o c o o o c o o e e Extrapolation of Hydrolysis Rates of Z-Methoxy- and Z-Ethoxy- Diethyl Acetal . . Extrapolation of S-Methoxy-Z-Pentanone Diethyl Ketal Hydrolysis Rate . . . . . . . Extrapolation of 5-Methylthio-2-Pentanone Diethyl Ketal Hydrolysis Rate . . . . . . . Ethanol Release by Hydrolyzing Z-Methyl- thioacetaldehyde Diethyl Acetal . . . . . . Ethanol Release Kinetics in the Hydrolysis of 2-Methylthioacetaldehyde Diethyl Acetal . Ethanol Release by Hydrolyzing S-Methylthio- Z-Pentanone DiethYl Ketal o o c c o e o o o Lysozyme Substrates . . . . . . . . . . . . 55 79 79 83 91 93 95 120 INTRODUCTION The elucidation of glycosidase activity may be approached from two directions; by study of the non-enzymatic hydrolysis of model glycosides or by study of the enzymatic hydrolysis process itself. Since glycosidase substrates are really cyclic acetals, much mechanistic information may be obtained from the hydrol- ysis rates of acetals and ketals. Very little has been said about the effect of nucleophiles on the hydrolysis rates of acetals and ketals, even though theoretical considerations indicate that nucleophiles should enhance the hydrolysis rate by stabilizing the carbonium-ion intermediate. One part of my thesis, then, will relate the prepara- tion and hydrolysis of acetals and ketals substituted with nucleophilic groups. Comparison of the hydrolysis rates will afford a mechanistic interpetation of nucleophilic effects in acetal and ketal hydrolysis, and by extrapolation, in glyco- sidase-catalyzed reactions. The second part concerns the problem of glycosidase catalysis in a more classical sense; it involves the prepara- tion of soluble, homogenous, chemically defined substrates for the somewhat atypical glycosidase, lysozyme (muramidase), and a study of the binding of these substrates to this enzyme, which catalyzes the cleavage of the 8(1e9) linkage between 2 N-acetyl muramic acid and N-acetyl glucosamine in bacterial cell wall polymers and the B(T*h) linkage in N-acetyl gluco- samine polymers (chitin). Investigation of the hydrolysis of acetals and ketals is not a recent phenomonon. In 1908, the hydrolysis of acetals was investigated by Fitderald and Lapworth.1 A 2’3’4'5 carried out a decade later, Skrabel and coworkers systematic investigation of the hydrolysis effects of acyl and aryl group substitutions. The following mechanism was postulated to account for the fact that the rate constants for alkyl-substituted acetal hydrolyses were constant: RCH(OX)2 + H20 g§if RCH(OX)(0H) + XOH k2 RCH(OX)(OH) -—9 RCHO + XOH Variations in the carbonyl portion of a series of pentaerythritol acetals were found to produce corresponding variations in the hydrolysis rates.6 Finally, Skrabel7 was able to demonstrate that carbonyl moeity alkyl substituent effects were additive. An investigation of the hydrolysis of cyclic acetals and ketals by Leutner8 demonstrated that acetal stability was a function of ring size; the 7-membered ring was most stable. Configurational analysis by Hermansg of acetal hydrol- ysis products indicated that the released alcohol retained its original configuration; therefore the carbon-oxygen bond of the carbonyl function, and not that of the alcohol function, 3 was cleaved. This mechanism was supported by the rate studies 10 on alkoxy- and hydroxy-substituted of Palomaa and coworkers ethyl acetals. Later work also supported this mechanism. Hydrolysis of the acetals of D-(+)-2—butanol11 and D-(+)-2- octanol12 produced no change in the final configuration of the alcohols. This retention of alcohol configuration led O'Gorman and Lucas12 to modify the alcoholic carbonium ion mechanism proposed by Hammett13 into a form which is accepted as the correct mechanism for acid-catalyzed acetal hydrolysis. Since the alcohol moeity was not racemized, the carbonyl car- bon atom, not the alcohol carbon atom, had to form the car- bonium ion: H R 3 +03 \C R\C'|' +ROH H/ on 11/ \OR H/ \on This mechanism was supported by the work of Zucker and Hammett14 which predicted that Specific oxonium ion catalysis should be a function of the Hammett acidity function Ho, rather than the concentration of the oxonium ion. Indeed, McIntyre and Longls'16 did demonstrate a direct relationship between the Hammett acidity function Ho and the hydrolysis rate of methylal in strong acid solutions. This indicated that the activated complex resembled the conjugate acid of methylal; the reaction was therefore unimolecular (no water molecules bound in the transition state). Support was also drawn from the observation that dimethyl acetal hydrolysis proceeded more rapidly in deuterium oxide than in water; LI, this was interpreted to indicate that a rapid protonation step was followed by a rate-determining decomposition of the complex.17 Kreevoy and Taft18’19’20 correlated inductive, resonance, and steric effects upon acetal hydrolysis and concluded that the transition state resembled a carbonium ion at the carbonyl carbon atom. The acid-catalyzed hydrolysis of glycosides presents an analogous situation: hydrolysis of several D-glucopyrano- sides in 018 enriched water indicated that the reaction pro- ceeded by hexose-oxygen bond cleavage. The center of substi- tution was therefore the C1 atom of the pyranose ring: the only exception to this rule is the hydrolysis of t-butyl-B- D-glucopyranoside which proceeds by alkyl-oxygen bond cleavage due to the favorable formation of the t-butyl car- bonium ion.21 However, glycoside hydrolysis is complicated by several possible hydrolytic mechanisms. One of these is a "bimolecular" synchronous mechanism with a linear transition state which leads to inversion of configuration: HO CHZOH CHZOH H HO OR H95” / H20 H 0+ The other mechanisms involve carbonium ion formation as in acetal hydrolysis, although the situation is complicated by the fact that the reaction may proceed with or without ring 22 opening: OH OH OHZOH OKOHD WE; p+H “fir Products.A C HZOH //,o H + ROH OH YHOR O CH OH H ‘§ 20H }1 +CHOR?r—9- Products B A?!" H The following transition state has been suggested in the hydrolysis of a substrate containing a nucleophile such as the N-acetyl group: CH OH 2 OH The actual mechanism of glycoside hydrolyses appears. to depend on the substrate and the reaction conditions. Thus the analogous-methanolysis of 2,3,h,6, tetra-O-methyl- d-D-glucopyranosyl chloride was unimolecular by kinetic analysis, but yielded an anomeric mixture predominating in either a- or 8- form, depending on whether or not chloride ion was present. On the other hand, the glucopyranosyl chloride reacted with thiophenoxide ion in propanol to yield the 8- product in a true bimolecular reaction. The analogous mannose compound underwent methanolysis to yield an anomeric 6 mixture which was unaffected by the presence of chloride ion, and which did not react at all with thiophenoxide ion. It appeared that the glucopyranosyl reaction mechanism could be either uni- or bimolecular: on the other hand the manno- pyranosyl system reaction mechanism appeared to be solely unimolecular due to steric effects. In general, the hydrolysis of simple glycosides appears to be unimolecular and to involve the formation of a carbonium ion in the rate determining step: that is, the rate appears to be dependent on HC rather than upon the concentration of oxonium ion.21 In all probability, these carbonium ions are ring-closed: the hydrolysis of a-methyléD-glucopyranoside was found to exhibit a primary oxygen isotope effect which was attributed to C1 carbon-oxygen bond cleavage during the rate-determining step. This is consistent with the formation of a ring-closed carbonium ion since the Cl-oxygen bond in the open-carbonium ion mechanism breaks in a step subsequent to the rate—determining step.25 It is notable that the effects of neighboring groups on the hydrolysis of acetals, ketals, and glycosides have been well documented only in the case of the carboxyl group. As early as 1919, Karrer26 noted that the B-D-glucoside of salycic acid (o-carboxyphenyl-B-D-g1ucoside) Spontaneously hydrolyzed in aqueous solution. This phenomonon was con- firmed by Helferich,27 who noted that spontaneous hydrolysis occurred only in unbuffered solutions and only when the glu- coside was in the un-ionized carboxyl form: the salt and 7 ester forms of this glycoside were inactive. Capon studied the anomalously rapid hydrolysis of o-carboxyphenyl-B-D- glucoside and postulated that the carboxyl group participated as a nucleophilic-Specific acid catalyst, a general acid 28 catalyst, or as a combination of the two mechanisms. How- 29 analyzed the scanty published data and con- ever, Bruice cluded that electronic effects upon simple Specific acid catalysis could account for the supposed carboxyl catalytic effect. Capon30 also studied the hydrolysis of a simple car- boxyl-substituted acetal and observed a rate enhancement over that eXpected for Specific acid catalysis (relative to the paggisomer and the methyl ester) and postulated the following mechanism: QED-CHZ-O-CHB OH fiHz e a(gums @8-00 + (1 Again Bruice29 analyzed the results and concluded that in this case, the water-deuterium oxide rate ratio indicated general acid catalysis or Specific acid-nucleophilic cataly- sis in a pH region in which only simple Specific acid catalysis should have been observed. Thus a carboxyl group effect may be operative in this instance. The hydrolysis of oligouronides also seemed to be influenced by intramolecular carboxyl group general acid 31 catalysis. 8 Bruice29 conducted a penetrating analysis of carboxyl group participation in ketal hydrolysis. He investigated the hydrolysis kinetics of 19 1,3 dioxanes and 1,3 dioxolanes: 7 of these were carboxyl-substituted. The ketal structures were chosen so that a differentiation between intramolecular general acid catalysis and Specific acid-nucleophilic attack could be made if carboxyl group participation was noted. Variations in the pH-log k profiles of carboxyl substi- obs tuted ketals indicated either undissociated carboxyl-group participation or simple specific acid hydrolysis of the anion form of these ketals. However, the carboxyl-substituted ketals exhibited no significant positive deviation from the Hammett plots of log kh versus 0, indicating that the reaction mechanism was simple specific acid catalysis. A water-deuterium oxide rate ratio of 0.20 also indicated Specific acid catalysis. -Thus the evidence for carboxyl group participation appears to be meager; the only probabke instances are the hydrolysis of the simple benzoic acid—substituted acetal30 and the hydrolysis of oligouronides.31 This fact is somewhat disappointing in view of the implication of carboxyl groups in the catalytic activity of 33 34 d-amylase,32 B-glucosidase, and lysozyme. 35 Lysozyme was discovered by Fleming in 1922; soon thereafter, a large number of lysozymes were characterized from diverse sources: 36 37 vertebrate tissues and secretions, invertebrates, 40 insects,38 bacteria,39 and even plants. These diverse 9 lysozymes, although differing slightly, have some common characteristics: They are basic proteins of low molecular weight (about 15,000), are stable at acidic pH (some even at 100° and pH 4.5 for 1-2 minutes), unstable at alkaline pH, and are capable of hydrolyzing M, lysodeikticus sus- pensions and solutions of N-acetylglucosamine polymers derived from chitin.36 Therefore lysozyme is also called B-glycosamidase, N-acetyl muramide glycanohydrolase or muramidase. The most common and readily available lysozyme is that derived from chicken egg white. This lysozyme may be crystallized directly from the egg white“0 and further 41 purified by chromatography on a Dowex-BO column. Hen egg white lysozyme is a Single polypeptide chain consist- ing of 129 amino acid residues whose sequence is knownu2'43 and whose structure has been established to 2 X resolution.44 The molecule is ellipsoidal, about 45 x 30 x 30 A, and possesses a marked cleft on one side which has been the object of much Speculation. This lysozyme has four disul- fide bridges, three io-residue c-helix runs, several turns of 3°010 helix and a section of antiparallel pleated sheet conformation: the remainder of the molecule has no well- defined conformation. Several active site amino acids have been detected by chemical means. Difference titrations of lysozyme versus lysozyme plus N-acetyl glucosamine or N-acetyl glucosamine trimer indicated a pK shift from 6.3 to 6.7 due to saccha— ride binding: the residue responsible for this perturbation 10 was thought to be Glu 35, since it apparently resides in a 45 relatively nonpolar environment. N-acetyl glucosamine polymers also perturb the lysozyme U.V. Spectrum“6 this Spectrum and that of free lysozyme“7 varies with pH to yield pK values resembling those derived from difference titra- tions.”5 X-ray diffraction perturbations caused by the binding of N-acetyl glucosamine, tri-N-acetylglucosamine, and N-acetyl glucosaminyl-N-acetyl muramic acid, and model building studies have indicated that the hexasaccharide composed of alternating N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) units is cleaved between residue 9 (NAM) and 5 (NAG).I+8 This was confirmed by the finding that the hexamer of NAG is cleaved into dimer and tetramer, this cleavage occurring two residues distant from the reducing end of the oligomer. The X-ray diffraction work also indicated that sub- strate binding was confined to the cleft of the molecule: the catalytically important residues appeared to be Glu 35 and ASp 52.48 Several catalytic mechanisms have been proposed22 which involve these residues. The first proposal is basi- cally the nucleophilic glycoside hydrolysis mechanism of Koshland.l+9 Glu 35 appears to be in a non-polar environ- ment and to be in a position to protonate the glycosidic oxygen atom: it may therefore assist in glycosidic bond rupture by general acid catalysis. On the other hand, Asp 52 appears to be in a polar environment and is probably 11 ionized: it may displace the disaccharide by nucleophilic attack on carbon C1 of the fourth hexasaccharide residue to form a glycosyl-enzyme which then undergoes nucleophilic attack by water (or other acceptor molecules). However, a detailed examination of the X-ray diffraction model of lysozyme made this mechanism an unlikely choice: the nucleo- phile and the leaving group do not appear to be able to attain a configuration favoring the required bimolecular transition state. This model also does not allow C1 and ASp 52 the proximity required for a covalent bond. An alternate mechanismz2 involves the formation of a stabilized carbonium ion in a manner analogous to carbonium ion formation in model system acid-catalyzed hydrolysis reactions. This mechanism invokes the following steps: 1. The substrate is attached to lysozyme and held in place by hydrogen-bond and hydrophobic interactions. The ring of residue 4 is distorted from the normal chair conformation into a half-chair conformation favoring carbonium-ion formation. 2. Glu 35, being un-ionized, protonates the glycosidic oxygen atom. 3. Heterolysis of the residue 4 C carbon-oxygen bond 1 yields a carbonium ion which is promoted and stabil- ized by the negative charge on ASp 52, which is in an ionic environment 3 X away. 4. The resulting free disaccharide diffuses out and a water molecule attacks the carbonium ion along the 12 same path with resultant overall retention of con- figuration at C1. Lowe advanced some interesting mechanisms for the lysozyme- catalyzed hydrolysis of o- and p-nitrophenyl B-D-chitobio- 50 sides which involve Glu 35 and ASp 52. One of these mech- anisms invokes participation of Glu 35 and the N-acetyl moiety of the terminal N-acetyl glucosamine residue with consequent formation of a cyclic ion. This mechanism again requires that the glucosidic ring undergoing hydrolysis take up a strained configuration, in this case a boat configura- tion. The other proposed mechanism postulated that Glu 35 acts as a general acid, while the carboxylate ion of ASp 52 acts as a nucleophile, yielding a glycosyl-enzyme intermedi- ate. It is therefore possible that some type of strong binding Situation, either ionic or covalent, obtains in lysozyme catalysis: nor should the possibility of neighbor- ing group assistance be overlooked. EXPERIMENTAL Reagents General All concentrated acids, common inorganic salts and organic reagents were reagent grade. Aceta1,gKetal, Tetrahydropyran ether model glycoside reagents) a. Benzene, anhydrous Reagent grade benzene was dried by azeotropic distil- lation. b. Bromoacetaldehyde diethyl acetal Matheson, Coleman and Bell reagent grade. c. Diethyl ether Mallinkrodt anhydrous ether. d. Diethyl malonate Matheson, Coleman and Bell reagent grade. e. 3,4 Dihydropyran Aldrich Chemical Co. practical grade was redistilled on a packed column: the fraction boiling from 83.4-84.5o was collected. f. Dimethyl formamide Eastman Kodak reagent grade was redistilled prior to use: the fraction boiling at 152-1540 was collected. 13 14 g. Dioxane Dioxane was purified by a modification of the method of Fieser.51 A solution of 2 1 dioxane, 200 ml water, and 27 ml concentrated hydrochloric acid was refluxed for 36 hr under nitrogen. The solution was cooled and shaken with potassium hydroxide pellets to saturation: the resulting upper dioxane layer was decanted and dried over potassium hydroxide pellets. The dried dioxane was refluxed with 2 g sodium borohydride for 24 hr prior to distillation directly from the borohydride. This purified dioxane was then refluxed with and redistilled from sodium borohydride immediately prior to use to remove any traces of peroxide formed during storage. h. Ethanol, absolute Gold Shield ZOO-proof ethyl alcohol was refluxed with and redistilled from barium oxide. 1. Ethanol assay system Determatube C-ALK ethanol assay system was purchased from Worthington Biochemical Company. J. Ethylene chlorohydrin Eastman Kodak reagent grade. k. Ethyl orthoformate Aldrich Chemical Company reagent grade. 1. 2-Ethoxyacetaldehyde diethyl acetal Aldrich Chemical Company and Matheson, Coleman and Bell. m. Methanol, absolute 15 J. T. Baker absolute methyl alcohol. n. 2-Methoxyaceta1dehyde dimethyl acetal Aldrich Chemical Company. 0. 2-(2-hydroxyethyl)-pyridine Aldrich Chemical Company. p. Thionyl chloride Matheson, Coleman and Bell. Lysozyme reagents a. Amberlite MB-3 mixed bed resin, 20-50 mesh Rohm and Haas b. Amberlite CG-120 resin Amberlite CG-120 type II (200 mesh and finer) pur- chased from Rohm and Haas had previously been classified for quantitative amino acid chromatography by the method of Hamilton:52 the fraction used residue which did not flow at min. c. Aminex AG SON-X2, 200-300 California Corporation BioRad Laboratories. d. Biogel P-4, 50-100 mesh BioRad Laboratories e. Carboxymethyl cellulose California Corporation f. Dialysis tubing in this work was the coarse a wash velocity of 590 ml per mesh, hydrogen form for Biochemical Research and for Biochemical Research. Cellulose "dialyzer tubing" (15/16 in), no. 4465-A2, 16 was purchased from A. H. Thomas Company and used in several substrate preparation experiments. However, this material did not retain lysozyme: therefore "Visking" sausage casing (16/32 in), purchased from Union Carbide Corporation, was utilized in dialyses when retention of molecules of m.w. greater than 10,000 was important. g. Dimethylsulfoxide Aldrich Chemical Company reagent grade. h. Dithiothreitol California Corporation for Biochemical Research. 1. Glass beads Minnesota Mining and Manufacturing Company "superbrite" type 130-5005 0.1 mm diameter glass beads were stirred in hot chromic acid cleaning solution for six hours, then exhaustively washed with distilled water and dried at 105°. J. Glycerol, anhydrous Reagent grade glycerol was distilled under reduced pressure (0.05 mm) and stored over phosphorus pentoxide. k. Guanidine hydrochloride Reagent grade guanidine hydrochloride (Mallinkrodt) was recrystallized from 95% ethanol or prepared by the addi- tion of concentrated hydrochloric acid to reagent grade guanidine carbonate (Eastman Kodak) to pH 7. The resulting solution was filtered, and evaporated to dryness: the off- white solid was recrystallized from 95% ethanol. 1. Hydroxylamine hydrochloride Matheson, Coleman and Bell practical grade. 17 m. Iodoacetic acid Eastman Kodak reagent grade. n. Lysozyme Worthington Biochemical Corporation twice recrystal- lized salt-free lysozyme and Pentex, Incorporated crystalline lysozyme. o. Micrococcus lysodeikticus cells, dried Sigma Chemical Company pfs grade p. Polychromatic ninhydrin spray solution This solution was prepared as described by Litwack.53 Solution A was a 0.2% ninhydrin solution made up in a mixture of 50 ml anhydrous ethanol, 10 ml glacial acetic acetic acid, and 2 ml 2,4,6 collidine. Solution B was a 1% solution of cupric nitrate trihydrate in redistilled water. Solutions A and B were combined in a 50:3 ratio (v/v) prior to use. q. Sephadex G-15 Pharmacia Fine Chemicals, Incorporated. r. Urea Mallinkrodt reagent grade. Urea solutions were deion- ized prior to use by storage over Amberlite MB-3 mixed bed resin. Preparations 2-Methylthioacetaldehyde The preparation of this aldehyde was a modification of the method of Cope and co-workers.54 A mixture of Z-methylthioacetaldehyde dimethyl acetal (20 ml), water (20 ml), and concentrated hydrochloric acid 18 (0.2 ml) contained in a 100 ml flask fitted with reflux con- denser and magnetic stirring bar was stirred for 30 min at room temperature, and then refluxed and stirred for 5 min to yield a homogenous solution. This solution was cooled and saturated with sodium chloride. The resulting organic layer was removed in a separating funnel (upper layer) and the water layer extracted 3 times with ethyl ether (15 ml por- tions). The ether extracts were combined with the organic layer: the resulting solution was dried over magnesium sul- fate. Filtration and distillation through a Vigereux column yielded a light yellow oil which was fractionally distilled on the same still at reduced pressure to yield 2-methylthio- acetaldehyde (3.8 g) b.p. 770 (105 mm). 2-Hydroxyethyl Methyl Sulfide This sulfide was prepared by a modification of the procedure of Windaus and Schildneck.55 Sodium (80.5 g) was reacted with absolute ethanol (1500 ml) in a 3-1 three-necked round bottom flask fitted with a stirrer, a reflux condenser, and a dropping fun- nel. When reaction had ceased, methyl mercaptan (supplied from a gas cylinder) was bubbled through the solution until it was saturated. The resulting sodium methyl mercaptide solution was heated to reflux. Heating was discontinued and ethylene chlorohydrin (302 g) was slowly added with vigorous stirring over a 2 hr period. Then 1000 ml of ethanol were 19 distilled off and the concentrate cooled and filtered. The precipitate was washed three times with the previously removed ethanol. The filtrate was fractionally distilled through a 50-cm packed column under reduced pressure to yield 2—hydroxyethyl methyl sulfide (283 g), b.p. 74° (20 mm). 2-MetMylthioacetaldehyde Diethyl Acetal The procedure employed was a modification of the method described by Cope.5u Sodium (6.9 g) was reacted with absolute Methanol (100 ml) in a round bottom flask fitted with a reflux con- denser and a drying tube. When the sodium methoxide solu- tion had cooled, a magnetic stirring bar and gas delivery tube were added to the flask. Methyl mercaptan (gas cylin- der) was slowly bubbled through the stirred solution to saturation. Then 2-bromoacetaldehyde diethyl acetal (60 g) was added and the resulting solution refluxed for 24 hr. Filtration and concentration by distillation on a Vigereux column still produced a suSpension which was again filtered; the filtered precipitates were washed with ether (10 ml) in each case. The resulting yellow ether solution was frac~ tionally distilled on a Vigereux column still to give 2- methylthioacetaldehyde diethyl acetal (20 g), b.p. 77.5-79.5° (13 mm). 20 2~Methoxyacetaldehyde Diethyl Acetal A solution of 2-bromoethanal diethyl acetal (30 g) and sodium methoxide (7.9 g sodium in 60 ml absolute meth- anol) was sealed into a heavy glass reaction tube and placed in a 1050 oil bath for 48 hr. The resulting reaction mixture was filtered and distilled to remove the methanol. Anhydrous ethyl ether (50 ml) was added to the Slurry, the resulting suSpension was filtered, and the precipitate washed with ether (25 ml). The filtrate was concentrated by distillation through a Vigereux column to yield a slurry of product and salt. Much of the remaining salt was removed by repetition of the ether-filtration-distillation step. The resultant yellow suspension was taken up in ether (60 ml) and centrifuged. The supernatant was distilled through a Vigereux column to remove the ether; the crude product was then distilled in the same still at reduced pressure to yield 2-methoxyacetal- dehyde diethyl acetal (4 g), b.p. 49.5-50.00 (12 mm). 4-Methylthio Butraldehyde Diethyl Acetal 2-ChloroetMyl methyl sulfide A slight modification of the method of Kerner and 56 Windaus was utilized to prepare this compound. A solution of thionyl chloride (68 g) in anhydrous chloroform (65 g) was added dropwise to a stirred solution of 2-hydroxyethyl methyl sulfide (50 g) in anhydrous chloroform (70 g). The 21 reaction mixture was heated once midway in the addition to maintain gentle chloroform reflux. Stirring was maintained for four hours after completion of the thionyl chloride addition. The chloroform was removed by distillation on a steam bath. The residue was distilled under reduced pres- sure to yield 2-chloroethy1 methyl sulfide (44 g), b.p. 48- 500 (22 mm). Ethyl-Z-metMylthio-ethyl malonate The preparation of this compound was a modification of the Organic Synthesis Ethyl-n-butylmalonate preparation.57 Diethyl Malonate (58 ml) was added dropwise over a period of one hour to a sodium ethoxide solution prepared by reacting sodium (8.33 S) with anhydrous ethanol (180 ml). The reaction mixture was then heated to 500 to prevent solidification and 2-chloroethyl methyl sulfide was added over a 2 hr period. The reaction mixture was refluxed for 2 hr, then cooled and filtered. The filtrate was distilled to remove the ethanol: the residue was washed once with 10% sodium chloride solution. The organic layer was removed and the water layer was extracted with ethyl ether (100 ml). The organic layer and the ether extract were combined and dried over anhydrous sodium sulfate. The ether was removed by dis- tillation at reduced pressure (22 mm): the residue was frac- tionally distilled at reduced pressure to yield ethyl-2- methylthio-ethyl malonate (40 g), b.p. 148-1500 (22 mm). 22 4—Methylthio-butyric acid Again, this preparation is a modification of the method outlined in qrsanic anthasis.58 Ethyl-Z—methylthio-ethyl malonate (40 g) was slowly added under nitrogen atmosphere to a hot (90°) solution of potassium hydroxide (40 g) in water (40 ml). This reaction mixture was refluxed for 3 hr: then water (40 ml) was added and 40 ml of liquid was distilled off to remove the ethanol. The remaining liquid was cooled and a cold solution of sulfuric acid (64 g) in water (70 ml) was added over a 1 hour period. The reaction mixture was refluxed for 8 hours and then cooled: the upper organic layer was removed and the water layer was extracted four times with ethyl ether (40 ml portions). The organic layer and ether extracts were combined and dried over anhydrous sodium sulfate. The ether was removed by reduced pressure distillation (15 mm) and the residue fractionally distilled at reduced pressure to yield 4-methylthio butyric acid (15.5 S) bop. 883900 (0060 mm). 4-Methylthiobutyric acid chloride Thionyl chloride (34.5 g) was slowly added to 4- methylthiobutyric acid (15.5 8). After 6 hours at room temperature, the residual thionyl chloride was evaporated at reduced pressure under nitrogen to yield 4-methylthiobutyric acid (10.5 s) b.p. 86-890 (10 mm). Elemental analysis: Calculated: c, 39.35% H, 5.91% Cl, 23.28% 3, 20.98% Found: C. 39-49% H. 6-09% Cl. 23-51% 5. 20-90% 39-35% 5.94% 23.44% 23 4-MetMylthiobutraldehyde Rosenmund reduction was utilized to prepare this alde- hyde. A solution of 4-methylthiobutyric acid chloride (10.5 g) in anhydrous benzene (50 ml) containing 5% palladium- barium sulfate catalyst (5 g) was heated to 700 and stirred while hydrogen was bubbled through the suSpension. The effluent gas was passed through 0.11 N sodium hydroxide solu- tion to monitor the progress of the reaction. When reaction was 80% complete by sodium hydroxide neutralization, the catalyst was filtered off and washed with anhydrous benzene (20 ml). The filtrate and washings were combined and the benzene distilled off at reduced pressure (15 mm) to yield a residue of crude yellow 4-methylthiobutraldehyde (10 g). Elemental analysis: Calculated: C, 50.08% H, 8.48% S, 27.10% Found: C, 50.92% H, 8.43% 3, 26.45% 26.34% 4-Methylthiobutraldehyde diethyl acetal The crude 4-methylthiobutraldehyde from the Rosenmund reduction (10 g) was mixed with triethyl orthoformate (25 g), absolute ethanol (10 ml) and ammonium chloride (0.1 g) and refluxed for 18 hours. Low boiling components, primarily ethyl formate, were stripped off during reflux on the Spin- ning band still. The ethanol was then distilled off and the residue fractionally distilled at reduced pressure the yield 4-methylthiobutraldehyde diethyl acetal (4 g) b.p. 102-1030 (10 mm). 24 5-Hydroxyvaleraldehyde 59 The method of Schniepp and Geller was used to pre- pare 5-hydroxyvaleraldehyde. Preparation of Valeraldehyde Diethyl Acetal A solution of valeraldehyde, (30 g), triethylorthofor- mate (60 g), absolute ethanol (10 ml) and concentrated sul- furic acid (0.05 ml) was kept at room temperature for 48 hr. The solution was then neutralized with ethanolic sodium ethoxide and fractionally distilled to remove ethyl formate and ethanol. Fractional distillation on a packed column at reduced pressure gave valeraldehyde diethyl acetal (28 g) b.p. 59° (15 mm). Z-Pentanone Diethyl Ketal A solution of 2-pentanone (43 g), triethylorthoformate (90 g), absolute ethanol (5 ml), and concentrated sulfuric acid (0.03 ml) was allowed to stand at room temperature for 36 hr. The resulting solution was neutralized with ethanolic sodium ethoxide, concentrated by fractional distillation on a Vigereux column, and fractionally distilled in the same still at reduced pressure to yield 2-pentanone diethyl ketal (51 g) b.p. 87-88° (14 mm). 25 Preparation of 5_Chloro-2-Pentanone A mixture of water (263 ml), concentrated hydrochloric acid (225 ml) and d-acetyl-Yubutyrolactone was heated to main- tain a rapid rate of carbon dioxide evolution. After 25 min, gas evolution had ceased: the heat input was then increased to rapidly distill off the ketone-water mixture. After 450 m1 distillate had been collected, 300 ml of water was added and distillation continued. This addition-distillation step was repeated: the distillates were combined and the ketone layer separated. The water layer was extracted three times with ether (100 m1 portions) and the extract combined with the ketone layer. After drying over calcium chloride, the ether was removed by distillation: the residue was fraction- ally distilled at reduced pressure to yield 5-chloro-2- pentanone (124 g) b.p. 59-600 (12 mm). The ketone was stored at 50 to retard decomposition. Preparation of 5-Chloro-2-Pentanone Diethyl Ketal A solution of 5-Chloro-2-pentanone (62 g), triethyl orthoformate (88 ml), absolute ethanol (5 ml) and concen- trated sulfuric acid (0.05 ml) was allowed to stand at room temperature for 24 hr. The red solution was neutralized with sodium ethoxide in ethanol and distilled to remove ethyl for- mate and ethanol. The residual oil was fractionally distilled at reduced pressure on a 1/2 by 20 inch column packed with 2 mm glass helices to yield 5~chloro-2-pentanone diethyl 26 ketal (74 g), b.p. 92-930 (13 mm). Elemental analysis: Calculated: C, 55.52% H, 9.84% Found: C, 58.86% H, 10.06% 58.63% 9.94% 5~Methylthio—2~Pentanone Diethyl Ketal A sodium methoxide solution prepared by reacting sodium (1.15 g) with absolute methanol (25 ml) was saturated with methyl mercaptan. The resulting sodium methyllnercap- tide solution was mixed with 5-chloro-2-pentanone diethyl ketal (10.7 8). sealed in a glass reaction tube, and placed in a boiling water bath for 24 hr. The reaction mixture was then cooled and filtered: the filtrate was concentrated by distillation, mixed with ether (10 ml) and again filtered. This filtrate was distilled at reduced pressure (13 mm) to yield the crude ketal which was fractionated on the Spinning band column at reduced pressure (13 mm) to yield impure ketal, b.p. 80—890. Refractionation of this material on a packed column at reduced pressure yielded 5-methylthio-2- pentanone diethyl ketal (5 g), b.p. 540 (0.025 mm), Elemental analysis: Calculated: C, 58.20% H, 10.75% 8, 15.54% Found: C, 58.63% H, 10.90% S, 16.47% 58.46% 10.69% 16.62% 27 5-Methoxy-2-Pentanone Diethyl Ketal A sodium methoxide solution prepared by adding sodium (1.15 g) to absolute methanol (25 ml) was mixed with 5- chloro-Z—pentanone diethyl ketal (10.7 g), sealed in a glass reaction tube and placed in a boiling water bath for 48 hr. The reaction mixture was then cooled, filtered, and concen- trated by distillation. Ether (10 ml) was added to the con- centrate and the precipitate removed by centrifugation. Removal of the ether by distillation afforded an oil, which when fractionally distilled at reduced pressure, yielded 5- methoxy-Z-pentanone diethyl ketal (4.5 g), b.p. 70-710 (13 mm). Elemental analysis: Calculated: C, 63.12% H, 11.66% Found: C, 63.33% H, 12.07% 2—Ethoxy Tetrahydropyran To a stirred solution of anhydrous ethanol (30 g) and concentrated sulfuric acid (0.1 ml) was added 3,4 dihydro- pyran (60 g). After 14 hr at room temperature, the reaction mixture was neutralized with ethanolic sodium ethoxide solu- tion and fractionally distilled at reduced pressure to yield 2-ethoxy tetrahydropyran (32 g), b.p. 40.5-41.00 (11 mm). 2-(2uMethoxyethoxy)-Tetrahydropyran Sixty grams of 3,4-dihydropyran were slowly added to a 28 stirred solution of 2-methoxyethanol (48 g) and concentrated hydrochloric acid (0.1 ml). The reaction mixture was kept at room temperature for 12 hr and then fractionally distilled at reduced pressure to yield 2-(2-methoxyethyl)-tetrahydro- pyran (43.5 8): b.p. 80-820 (15 mm). 2-(2eMethylthioethoxyleTetrahyerpyran Thirty-two grams of 3,4-dihydropyran were slowly added to a stirred solution of 2-methylthioethanol (30 g) and concentrated hydrochloric acid (0.1 ml). The reaction mixture was kept at room temperature for 12 hours and then fractionally distilled at reduced pressure to yield 2-(2- methylthioethoxy)-tetrahydropyran (25 g), b.p. 114-1160 (15 nun). 2-(2mPyridinylethoxy)-Tetrahydropran Hydrogen chloride was bubbled into a solution of 3,4- dihydropyran (16.8 g), 2—(2~hydroxyethyl)-PYridine (24.6 g) and dimethylformamide (30 m1) contained in an ice-cooled round-bottom flask fitted with a drying tube and reflux con- denser until 6 g of HCl had been added. The solution was then heated to 900 and held at that temperature for 4 hr, then cooled and held at room temperature for 3 hr. The resulting brown solution was neutralized with barium oxide for 12 hr, suspended in ethyl ether (200 ml) and centrifuged to yield a yellow solution. Concentration by evaporation at 29 reduced pressure (11 mm), followed by distillation at reduced pressure (11 mm) afforded a brown oil. Fractional distillation at reduced pressure on a Vigereux column still yielded 2-(2-pyridinylethoxy)-tetrahydropyran (9.5 g), b.p. 84-870 (0.75 mm). Elemental analysis: Calculated: C, 69.51% H, 8.25% N, 6.76% Found: C, 69.07% H, 8.07% N, 7.16% Preparation of Acetate Buffers for Ketal Hydrolysis Rate Determinations The ionization constant of acetic acid in 49.6% dioxane-~50.4% water (by weight), the final composition of the ketal hydrolysis medium, was determined by extrapolation from the acetic acid ionization constant data published by Harned and Owen:60 it was found to be 2.63 x 10-7M (pK = 6.58). Acetate buffers were prepared by weighing the required amounts of glacial acetic acid and sodium bromide (when required to adjust ionic strength) into a volumetric flask. The required amount of 3.030N sodium hydroxide was then pipetted into the flask and the contents diluted to the mark to yield the buffer. A Beckman Model G pH meter was used to determine the pH values of the solutions. Table I summarizes the preparation of the acetate buffers. The concentrations listed are those found prior to dilution with dioxane: the actual hydrolysis concentrations are one-half the stated concentrations. The notation (H+) 30 TABLE I Preparation of Acetate Buffers for Ketal Hydrolysis Rate Determinations The figures in parentheses indicate the grams of glacial acetic acid and sodium bromide per liter buffer or the milliliters of 3.03ON sodium hydroxide per liter buffer. Acetic Sodium Sodium 7 7 + pH Acid, M Acetate, M Bromide, M F 10 (H+) 10 (H )c 4.18 0.0087 0.0025 0.0000 0.0050 9.14 11.8 (0.676g) (0.824ml) 4.40 0.0500 0.0250 0.0000 0.0500 5.26 10.3 (4.502g) (8.24ml) 4.64 0.0500 0.0500 0.0000 0.1000 2.63 6.45 (6.005g) (16.65ml) 4.22 0.0750 0.0250 0.0250 0.1000 7.89 19.2 (6.0053) (8.30ml) (2.569g) 4.34 0.1516 0.0744 0.000 0.1588 5.26 12.0 4.20 0.0750 0.0250 0.0750 0.2000 7.89 24.3 (6.0058) (8.27ml) (7.7308) Dilution of the pH 4.22 and pH 4.64 acetate buffers with 0.0500 M sodium bromide (10.291 g per 2 1) solution by factors of 1:2 and 1:10 produced diluted buffers of identi- cal ratios, ional strengths and hydronium concentrations. 31 refers to the uncorrected hydronium ion concentration of the dioxane-diluted buffer: (11+)c refers to the hydronium ion activity of the dioxane-diluted buffer corrected for secon- dary salt effects by the use of the following equation:61 (HI)C = K (acid) ft (salt) ft is determined by use of: ft = 1°26’r1 r1: Ional strength 1 + 2.0JF‘ Determination of Hydrolysis Rates All acetal and ketal and most tetrahydropyranyl ether hydrolysis rate determinations were based on increments in absorbance created by the n-——»n* transition of the car- bonyl group of the aldehyde or ketone produced upon hydrolysis. Although the aldehyde or ketone produced in the course ofhydrolysis becomes hydrated, the equilibrium between non- hydrated and hydrated forms apparently is very rapid since kinetic measurements are not perturbed by this phenomonon. This situation also appears to apply to the equilibrium between 5-hydroxyvaleraldehyde and its cyclic hemiacetal. All kinetic measurements were carried out in solutions of 50% dioxane - 50% perchloric acid or buffer solution (by volume) this correSponds to a 49.6:50.4 weightzweight ratio.6 Absorbance was measured in a Beckman D.U. or Cary Model 11 Spectrophotometer: In all cases the cell compartment was thermostatted to 25.0 t 0.05°. 32 The concentration of acetal, ketal, or tetrahydropyran ether was adjusted so that the absorbance of the completely hydrolyzed solution was less than 1.0. In most cases, absorbance measurements were carried out in 1 cm quartz cells. However, the low solubility of several of the compounds necessitated a lower concentration and measurement in a 10 cm quartz cell (Cary 11). Changes in absorbance (increments) were read at fixed time intervals on the Beckman DU or read off the chart at fixed intervals in the case of the Cary 11. The log10 of these increments was then plotted as a function of time in seconds. The slope of this line multiplied by the natural log conversion factor 2.303 then gave the value of the first-order hydrolysis rate constant k The pseudo-second 1. order hydrolysis rate constant was determined by dividing the first-order rate constant by the acid concentration in the case of perchloric acid-catalyzed hydrolyses, or by the hydronium ion activity in the case of the buffer-catalyzed hydrolysis runs. The hydronium ion activity of the buffer solutions was calculated according to the following formula: (3+) = k (acid) - (salt) rid f+ is determined from the relationship: f ____ 1.26/P i 1 + 2.07[1 wherelfi is the ional strength of the buffer solution. 33 Certain of the acetals and ketals exhibited a primary salt effect: In these cases the hydrolysis rates were extra- polated to zero ional strength. Procedure for Determination of Acetal, Ketal,_and Tetrahydropyran Mydrolysis Rates Hydrolysis reaction mixtures were prepared by two methods. The first method employed two flasks immersed in a 25.00 water bath to equilibrate the dioxane (5-15 ml) acetal/ ketal/tetrahydropyran solution and the perchloric acid or buffer solution. At zero time an equivalent (5-15 ml) volume of perchloric acid or buffer was rapidly pipetted into the dioxane-acetal/ketal/tetrahydropyran solution and mixed by shaking. The reaction mixture was then transferred to a 1- or 10—cm quartz cell: the filled cell was placed in a thermo- statted Spectrophotometer compartment (25.00) and the absor— bance determined as a function of time. The second method employed a reaction flask consisting of an inner well surrounded by a concentric outer well. The appropriate amounts of acetal, ketal or tetrohydropyran and dioxane (5-15 ml) were pipetted into the center well: the perchloric acid or buffer was then carefully pipetted into the outer well. The flask was equilibrated in a 25.00 water bath. The solutions were very rapidly mixed at zero time by violent shaking: the reaction mixture was transferred to a 1 or 10 cm quartz cell. The cell was placed in a thermostatted spectrophotometer compartment (25.00) and the absorbance 34 determined as a function of time. Procedure for Determination of Tetrahydropyran Hydrolysis Rates Since the high absorbance of the pyridine ring system of 2-(2-pyridinyl-2-ethoxy)—tetrahydropyran effectively blanked out that of the aldehyde produced by acid-catalyzed hydrolysis, rate measurement by absorbance increments was not possible. Therefore the hydrolysis rates of several tetra- hydropyran ethers were followed by a modification of the 65 The hydrogen peroxide assay of Satterfield and co-workers. assay is based on alkaline hydrogen peroxide oxidation of liberated aldehyde: the amount of acid formed is determined by back-titration with hydrochloric acid. The hydrolysis reaction mixture was prepared by weigh- ing 203-217 mg 2-(2-pyridinylethoxy)-tetrahydropyran or 155- 167 mg 2-(2-methoxyethoxy)—tetrahydropyran into the center compartment of a reaction flask. Perchloric acid or acetate buffer was pipetted into the outer compartment. The flask was equilibrated in a 25.00 water bath: the contents were mixed at zero time by shaking in the bath. The reaction was allowed to proceed at 25.00. At 100- or ZOO—sec intervals a 2 ml portion was withdrawn and quenched in 20 ml of 0.02 N sodium hydroxide solution. A solution of 3% hydrogen peroxide (2 ml) was added to each flask. The flasks were then placed in a 60° water bath for 5 minutes, then cooled and titrated with 0.01 N hydro- 35 chloric acid to a phenolpthalein end point. (the amount of perchloric acid present in the reaction mixture aliquot was subtracted). The assay was standardized with known quantities of the tetrahydropyran ether hydrolysis product, 5-hydroxy- valeraldehyde (1 x 10-5, 5 x 10'5, and 10 x 10'”5 moles). First order rate constants were determined by two methods. Several constants were determined by plotting aldehyde concentration as a function of time. The half-life of the reaction as determined from this plot then allowed a determination of the first order rate constant k1 by the fol- lowing relationship: Most of the rate constants were determined by plotting the logarithm of the fraction Ct/Co (Ct is the concentration of unhydrolyzed tetrahydropyran ether at time t and Co is the concentration at zero time). The slope of this line multi- plied by the natural logarithm conversion factor 2.303 than yielded the first-order hydrolysis rate k1. In both cases, the pseudo-second-order hydrolysis rate constant k was obtained by dividing k by the acid 2 1 concentration. Rate of Ethanol Formation in HydrolyzingSamples of Methylthioacetals and Ketals The rate of ethanol formation in hydrolyzing samples 36 of 2-methylthioacetaldehyde diethyl acetal or 5—methylthio- 2-pentanone diethyl ketal was determined by quenching portions of the hydrolysis solution. The alcohol content of the quenched samples was determined by a coupled enzyme system in which the horse liver alcohol dehydrogenase mediated reduction of NADH was coupled to methylene blue reduction (decolorization) by a diaphorase from Clostridium kluyyeri (Determatube C-ALK). 2-Methylthioacetaldehyde diethyl acetal A solution of 9.6—12.3 mg of 2-methylthioacetaldehyde diethyl acetal in 10.0 ml 0.020 M hydrochloric acid was pre- pared by equilibrating acetal and acid separately in a 25.00 water bath: the two components were mixed at zero time and allowed to hydrolyze at 25.00. At appropriate intervals, usually 100 seconds, 1.0 ml samples were withdrawn from the reaction solution and quenched in 10.0 ml 0.02 M sodium bicarbonate solution. The quenched samples were then assayed for ethanol concentration by mixing 0.1 ml of each sample with 3.0 ml of the standard C-ALK assay system in a 1 cm cuvette. The absorbance drop at 600 mu allowed a determination of the ethanol concentration: a blank consisting of 0.1 ml of a solution of 12 mg 2-methylthio- acetaldehyde in 10 ml water and an ethanol standard containing 2.0 ug ethanol per 0.1 ml allowed an accurate determination of the ethanol concentration in the samples. Ethanol evolu- tion was plotted as a function of time (Figure 7). Ethanol release kinetics were determined by plotting ethanol concen- tration increments as a function of time (Figure 8). 37 5-MetMylthio-2-pentanone diethyl ketal A solution of 90.7-97.5 mg of 5-methylthio-2-pentanone diethyl ketal in a mixture of 5.0 ml dioxane and 5.0 ml 0.1 M pH 4.34 acetate buffer was prepared by equilibrating ketal and dioxane buffer solution separately in a two-compartment reac- tion flask in a 25.0° water bath. At zero time the two com- ponents were rapidly mixed and the resulting solution allowed to hydrolyze at 25.00. At appropriate intervlas, usually 600 seconds, 0.1 ml samples were withdrawn and quenched in 5 ml (first two samples) or 10 ml (remaining samples) of 0.002 M sodium bicarbonate solution. The quenched samples were assayed for ethanol con- centration by mixing 0.1 ml of each sample with 3.0 ml of the standard CqALK assay system in a 1 cm cuvette. The ethanol concentration was derived from the decrease in absorbance at 600 mu with the aid of an ethanol concentration curve prepared from standard solutions containing 1, 2, and 3 ug ethanol per 0.1 ml. The assay response was found to be linear with alcohol concentration. The assay blank consisted of 0.1 ml of a mixture of 0.1 ml dioxane buffer solution and 10 ml of the bicarbonate solution. Ethanol evolution was plotted as a function of time (Figure 9). Preparation of Micrococcus Lysodeikticus Cell Walls Cell walls were prepared from dried M, lysodeikticus cells by a modification of the method of Sharon and Jeanloz.66 38 M, lysodeikticus cells (15 g), "Superbrite" 0.1 mm glass beaks (250 g) and redistilled water (250 ml) were placed in a 400 ml stainless steel Sorvall Omni-mixer assembly. The assembly was immersed in an ice-salt bath in the cold room to equilibrate: The cold mixture was then homogenized at full mixer Speed for 50 minutes. The resulting suSpension was decanted from the glass beads into an ice-cooled 2-1 beaker. The beads were washed with five 200 ml portions of ice-cold redistilled water and the washings combined with the previously decanted suspension. The suSpension was centrifuged at 3000 rpm for 20 minutes at 0° (all centrifugations were carried out in a Sorvall RC-2B centrifuge equipped with a GSA head). The precipitate was discarded: the supernatant was centrifuged at 10,000 rpm for 20 minutes at 0° to sediment the cell walls. The cell walls were washed three times by suSpension in 400 ml portions of ice-cold redistilled water followed by centrifugation at 10,000 rpm for 20 minutes at 00. The washed cell wall pellet was suSpended in redistilled water (400 m1) and centrifuged at 3000 rpm for 20 minutes at 0°: the pellet, consisting of residual heavy contaminants, was discarded, the supernatant was centrifuged at 10,000 rpm for 20 minutes at 0°. The cell wall pellet was suSpended in redistilled water (250 ml) and held at 100° for 20 minutes in a boiling water bath. The boiled suspension was centrifuged at 10,000 rpm for 20 minutes; the pellet was lyophilized to yield 39 off-white amorphous cell wall material (2.15 g). Hydrolysis of M. Lysodeikticus Cell Walls Hydrolysis yieldingghexa-. tetra-, and disaccharides and gMycopeptide I This hydrolysis of cell walls is an adaptation of the method of Sharon 2; 3;.67 Dry M, Mysodeikticus cell wall (2.1 g) was suSpended in 0.05 M ammonium acetate solution (100 ml): Pentex three times.recryStallized lysozyme (5 mg) and toluene (0.3 ml) were added and the suspension held at 37° for 24 hr. The resulting mixture was transferred to a 50 cm dialysis bag formed from 18/32 inch Visking sausage casing. The bag was suspended in a vertical length of 21 mm diameter glass tubing fitted with inlet and outlet tubes: dialysis was carried out by passing 600 ml toluene-saturated redistilled water through the glass tubing over a 12 hr period. Concentration of the dialysate on a rotary evapor- ator followed by freeze drying yielded 450 mg of yellow solid. This material was dissolved in 5 ml water and placed on a 3.5 x 20 cm Amberlite CG-120 column in the acid form. The column was then eluted with 500 ml of redistilled water: concentrating the eluate on a rotary evaporator and freeze drying the concentrate produced 210 mg of white solid. 40 Hydrolysis lieldinghexasaccharide and glypopeptide GP-l A suspension of M, Myggdeikticus cell walls (4.00 g) and toluene (1.0 ml) in an 0.050 M ammonium acetate solution 200 ml) containing Pentex lysozyme (8 mg) was magnetically stirred for 12 hr at 37°. The resulting yellow solution was placed in 15/16 inch dialysis tubing and dialyzed against redistilled water (700 ml) for 24 hr. The dialyzate was lyophilized to yield a yellow powder (350 mg). The lyophilized powder was dissolved in redistilled water (5 ml) and applied.to an Amberlite CG-120 HI column. The column was eluted with redistilled water (600 ml) at a rate of 100 ml per hr: the eluate was lyophilized to yield a white amorphous powder (284 mg). Hydrolysis yielding_glycgpeptide.§§:M§ M, lysodeikticus cell walls (1.7 g) were suSpended in 0.05 M ammonium acetate solution (100 m1): Worthington lysozyme (4 mg) and toluene (0.3 ml) were added and the sus— pension held at 37° for 2.5 hr. Then carboxymethyl cellu- lose in the sodium form (15 g) and redistilled water were added and the suSpension stirred for 1 hour at room tempera- ture: additional carboxymethyl cellulose (15 g) was added and stirring was continued for 2 hr. The thick suspension was diluted by the addition of redistilled water (100 ml) and centrifuged at 10,000 rpm for 20 min: the pellet was washed by resuSpension in redistilled water (100 ml) and centrifugation at 10,000 rpm for 20 min. The supernatants 41 were combined and lyophilized to yield an off-white powder (1027 8); Automated Gel Filtration Apparatus An automated gel filtration apparatus, which is dia- grammed in Figure 1, was utilized to fractionate the lyxo- zyme M, ;ysodeikticus cell wall digests. A Milton Roy "Minipump" metering pump pumped boiled redistilled water (via 1 mm id polyethylene tubing) through a 95 cm bed of Sephadex G-15 contained in a 100 x 2.5 cm Kontes chromatography column at a rate of 10 ml per hour. The inner surface of the column was coated with dichlorodi- methylsilane to reduce interface effects and improve resolu- tion. The top cap of the column was fitted with a stopcock so that air could be purged from the system prior to use. The bottom cap contained a stopcock so that the system could be closed off when it was not in use. The column eluate then flowed through 0.05 mm poly- ethylene tubing to a 1 cm path-length flow cell mounted in the sample compartment of a Beckman DB spectrophotometer. The monitored effluent then passed through 0.05 mm id poly- ethylene tubing to a Technicon fraction collector, where it was drop-counted into 3 ml fractions. The Spectophotometer output was fed into a Sargent SRL recorder fitted with log conversion gears and a 1 inch per hour chart motor. The recorder was equipped with an event marker connected in series with the fraction collector 42 Figure 1: Diagram of automated gel filtration apparatus. The system utilized a Sephadex G-15 column (2.5 x 95 cm), a Milton Roy "Minipump" metering pump, a Technicon frac- tion collector, a Beckman DB Spectrophotometer, and a Sargent SRL recorder fitted with an event marker. 43 AUTOMATED GEL FILTRATION APPARATUS saws»;- . II “‘1 Sephadex Sargent SRL . ' c-Is I Column A. Recorder : II J I f—_ I I i I i I . I I I I I I I r l ' I r _<, I ' * I A I LJJ Beckman DB : sh t- “ 80%fo $1.32.; I! Spectrophotometer : - I I I II . I I r 4 I Fraction ,, Metering I Eluent : CoIIector Pump . : M— | I...- sxeri--.,- resist. .. - .-s_i.93.oi---.,_I Figural 44 motor so that the absorbance peaks could be correlated with fraction numbers. Since the M, lysodeikticus cell wall saccharides absorb strongly in the 220-240 mu region, a plot of A220-240 as a function of time as provided by the recorder indicated saccharide elution peaks without recourse to lengthy tube- by-tube analyses. Preparation of Disaccharide,gTetrasaccharide. and "Fraction 1" Hexasaccharide-Glyoopeptide Mixture A 100 mg sample of M, lysodeikticus cell wall saccha- ride mixture (24 hr digest) was dissolved in 2 ml of redis- tilled water and placed on the Sephadex G-15 column of the automated gel filtration apparatus. The column was then eluted with boiled redistilled water at a rate of 10 ml/hr. Absorbance was measured at 230 mu: the recorder trace showed six distinct peaks (Figure 2). Peaks 1, 2, and 3 when lyophilized yielded useful amounts of material (Table II): fractions 4, 5, and 6 yielded minute quantities of material. Descending chromatography of the first three fractions was carried out on Whatman No. 1 paper for 50 hr in n-butanol/ acetic acid/ water (25/6/25 v/v, upper phase). N-acetyl glucosamine was used as a standard since the solvent was run off the end of the paper: Spot concentrations were 100 pg. Chromatograms were visualized by the method of Sharon and Seifter:°° chromatograms were dipped into a 0.5 M solution 45 Figure 2. Automated Gel Filtration of M, lysodeik- Eiggg Saccharides. A 100 mg load of 24-hour digested M, lysodeikticus saccharides was placed on the 2.5 x 95 cm Sephadex G-15 column of the automated gel fil- tration apparatus and eluted with boiled redistilled water at 10 ml/hr. Peaks 1, 2, and 3 were reapectively hexasaccharide-glycopeptide mixture, tetrasaccharide and disaccharide. Peaks 4, 5, and 6 yielded amounts too small to be characterized. 46 AUTOMATED GEL FILTRATION OF LA. lysodeikticus SACCHARIDES 1.00— l I I I I I I In 0.80 ‘- < 0.60 - an 8 . m 0-40— ‘° I < 0.20- 0.00 -~ 6 5 4 . 3 2 "I\___ l I l I ' L I I ' 120 no 100 90 80 70 60 F R ACT ION NUMBER Figure 2 47 TABLE II Sephadex G-15 Gel Filtration of M. lysodeikticus Cell WallSaccharides The column load was 100 mg of M, Mysodeikticus cell wall saccharide mixture: fractions containing a peak were pooled and lyophilized. Peak volume refers to the volume required to elute a peak completely: fraction volume refers to the volume pooled and lyophilized to form a fraction. -_ L L ‘ J _,__ .J— Lyophilized Fraction Peak Volume, ml Fraction Volume, ml Weight, mg 1 36 27 7.1 2 36 18 27.5 3 45 24 44.7 48 of sodium hydroxide in a mixture of ethanol and n-propanol (6/4, v/v) and then were placed in a 1200 oven for 5 to 10 minutes. Chromatogram Spots could then be detected by their yellow fluorescence under short-wavelength ultraviolet light. Fraction 1 produced no well-defined spot: some material was retained at the origin, while the remainder formed an ill-defined streak. Amino acid analysis of Fraction 1 (Table III) indicated that it was a mixture of hexasaccharide and glycopeptide of nearly equal molecular weight. Fraction 2 produced a well-defined Spot of R8 (mobility relative to n-acetyl glucosamine) = 0.47-0.54, which indicated that it was the tetrasaccharide: N-acetyl-B-D-glucosaminyl- (1e4)-N-acetyl-B-muramyl-(1+4)~N-acetyl-B-D-glucosam1nyl- (144)-N-acetyl muramic acid1 (muramic acid is the trivial name for 2—amino-3-O-ID—1-carboxyethyl)42-deoxy-D-glucose). Ele- mental analysis of fraction 2 and determination of water of hydration by weight 1083 upon dehydration at 78.5° over phos- phorus pentoxide in an evacuated (0.005 mm) Abderhalden drying pistol indicated that the lyophilized product approximated the composition of an octahydrate: Weight loss: Calc. 12.88%, Found 13.0%. Elemental analysis: Calculated: C, 40.79% H, 7.03% N, 5.01% Found: C, 40.97% H, 6.86% N, 4.91% Amino acid analysis of fraction 2 hydrolysates (constant boiling HCl, 105°, 24 hr) yielded equimolar quantities of TABLE III Amino Acid Analysis of Fraction 1 Samples (3 mg) of Fraction 1 were hydrolyzed (constant boiling HCl, 105°, 24 hr) and subjected to amino acid analysis on the Spinco Automatic Amino Acid Analyzer. Muramic acid and glucosamine values were corrected for loss incurred during 24 hr hydrolysis at 105°. The presence of histidine is an indication of cell wall contamination by cellular components. All values are expressed in umoles/mg fraction 1. Muramic ' Expt. Acid Glucosamine Lys His Glu Gly Ala 391 1.7 1.5 0.11 0.07 0.14 0.15 0.30 396 1.8 1.7 0.24 0.04 0.27 0.28 0.58 50 muramic acid and glucosamine (corrected for hydrolysis loss) and very minor quantities (less than 0.01 umole) of lysine, histidine, glycine, alanine, and glutamic acid. Digestion of fraction 2 by lysozyme (4 mg Fr. 1 and 0.1 mg Worthington lysozyme in 0.2 ml 0.05 M ammonium acetate solution held at 37° for 24 hr), followed by chromatography as previously described yielded a vivid spot, R8 = 1.0 and a very faint spot, R8 = 0.50, corresponding to the original tetrasaccharide. Fraction 3, when chromatographed as previously described, produced a well-defined spot, R8 = 0.96-1.0, indi- cating that this fraction is the disaccharide N-acetyl-BAD- glucosaminyl-(1e4)-N-acetyl-muramic acid (R8 = 1.0). Amino acid analysis of fraction 3 hydrolysates (constant boiling H01, 105°, 24 hr) yielded equimolar quantities of muramic acid and glucosamine and insignificant quantities of lysine, histidine, glycine, alanine, and glutamic acid (less than 0.01 umole). Chromatography of fractions 4, 5 and 6 gave no fluores- cent Spots: amino acid analysis of hydrolysates of these frac- tions (constant boiling HCl, 105°, 24 hr) indicated the pres- ence of ammonia and minute quantities of glucosamine, muramic acid, lysine, glycine, alanine, and glutamic acid. Preparation of M. lysodeikticusCell Wall Hexasaccharide and Glycopeptide GP-i Three 100 mg portions of the 12 hour lysozyme-digested “J .__a 'ri rn 51 M, Mysodeikticus cell wall saccharide preparation were suc- cessively fractioned by dissolving the portions in 2 ml redistilled water and placing the resulting solutions on the automated gel filtration apparatus. The column flow rate was 10 ml/hr, the absorbance was measured at 230 mu, and 3 ml fractions were collected. Fractions 1, 2, and 3 corresponding to the first three peaks, were collected and lyophilized from each run. The lyophilized fraction 1 preparations were then pooled to yield 64 mg of tan powder. This material was then fractionated by a modification of the glycopeptide separation method of Mirelman and Sharon.69 The pooled fraction 1 material was dissolved in 2 ml redistilled water, and the pH of the solution adjusted to 2.3 with formic acid. The solution was then placed on a 40 x 1.5 cm column of BioRad Aminex AG 50qu2 resin which had previously been equilibrated with 0.2 M pH 2.9 pyridine- formic acid buffer. The column was first eluted with 50 ml of 0.2 M pH 2.9 pyridine-formic acid buffer at a rate of 50 ml per hr: elution was continued with a 500 m1 gradient from 0.2 M, pH 2.9 to 1.0 M pH 5.3 PYridine-formic acid buffer (250 ml of each buffer). The column eluate was collected in 5 ml fractions. Fractions were analyzed by evaporating selected tubes to dryness on the rotary evaporator. Redis- tilled water (3 ml) was added to each tube and the absorbance of the resulting solutions was measured at 240 mu. Two A240 peaks were visible: one at an elution volume of 35 ml (A-i), the other at an elution volume of 200 m1 52 (A-Z). Fractions corresponding to these peaks were pooled and lyophilized to yield 25 and 31 mg reSpectively. Samples (3 mg) of fractions A-1 and A-2 were subjected to hydrolysis (constant boiling RC1, 105°, 24 hr) and amino acid analysis: the results are summarized in Table IV. Fraction A-i, when chromatographed on Whatman No. 1 chromato- graphy paper in i-butanol/acetic acid/water (25/6/25, v/v, upper phase) for 48 hr and visualized as previously described, yielded a fluorescent Spot, R8 = 0.21 (mobility relative to N-acetyl glucosamine). Fraction A-i therefore appears to be the hexasaccharide, N-acetyl-B~D-g1ycosaminyl-(1»4)-N-acetyl- B-muramyl-(1~4)-N-acetyl-B-D-glucosaminyl-(1e4)-N-acetyl-B- muramyl-(1‘4)-N-acetyl-B-D-glucosaminyl-(1»4)-N-acetyl muramic acid. Fraction A-2, when chromatographed on Whatman No.1 paper in 1-butanol/acetic acid/water (4/1/5, v/v, upper phase) with an alanine standard for 48 hr, formed a well- defined Spot, Rala = 0.10 (mobility relative to alanine). The amino acid composition and chromatographic behavior of this glycopeptide (GP-1) indicated that it was identical with the glycopeptide GP-i isolated by Mirelman and Sharon.69 Preparation of M. lysodeikticus Cell Wall Glycopeptide GP-2 Portions (200 mg) of the carboxymethylcellulose- treated lysozyme M, lysodeikticus cell wall digest were 53 dissolved in 2 ml portions of water and placed on the column of the automated gel filtration apparatus. The Sephadex column was eluted with boiled redistilled water at a rate of 12 ml per hr: the eluate was monitored at 240 mu and col- lected in 5 ml fractions. The A240 trace exhibited one large symetrical peak at an elution volume of 140 ml (gel-filtered fraction 1 consis- tantly elutes at this volume). Very minor disaccharide and tetrasaccharide peaks were observed in this instance. This peak was collected by pooling the fractions under the peak: freeze drying this solution yielded 70-90 mg of white amor- phous powder. A 250 mg sample of fraction 1 pooled from several gel filtration runs was dissolved in 5 ml of redistilled water. The pH of the solution was adjusted to 2.2 with formic acid: the solution was then placed on a 75 x 2.5 cm column of Aminex AG 50W-X2 resin which had previously been equilibrated with 0.2 M pH 2.9 pyridine-formic acid buffer. The column was first eluted with 200 ml of 0.2 M pH 2.9 Pyridine-formic acid buffer and then switched to a 1000 ml gradient from 0.2 M pH 2.9 to 1.0 M pH 5.3 PYridine-formic acid buffer (500 ml of each buffer). The column eluate was collected in 5 ml fractions. The fractions were analyzed by concentrating selected tubes on a rotary evaporator: residual pyridine was removed by evaporation MM.y§ggg (0.025 mm) over concentrated sulfuric acid. 54 Redistilled water (5 ml) was added to each of these tubes and the absorbance of the resulting solutions was measured at 240 mu. The A240 plot formed one large symmet- rical peak at 115 ml elution volume (Figure 3): no other well-defined, large peaks were found. The fractions contain- ing this peak were pooled and lyophilized to yield 90 mg of amorphous white powder. Hydrolysis (constant boiling HCl, 105°, 24 hr) of a sample (4 mg) of this glycopeptide material (GP-2) followed by amino acid analysis of the hydrolysate yielded results summarized in Table IV. Chromatography of this glycopeptide fraction, utiliz- ing alanine as a reference, on Whatman No. 1 paper in 1-butanol/acetic acid/water (4/1/5, v/v, upper phase) for 48 hr yielded a Single Spot of Ra a = 0.22. This chromato- 1 gram was developed by two successive methods: the first method utilized the chromogenic ninhydrin Spray. The chro- matogram was sprayed with the solution and then placed in a 110° oven for 15 minutes. A well-defined alanine Spot and a very faint glycopeptide spot were noted. The same chro- matogram was then dipped in 0.5 M sodium hydroxide solution (6:4 ethanol-propanol) and placed in a 120° oven for 15 minutes. A faintly fluorescent spot was noted under ultra- violet light: it was in the same position as the ninhydrin- glycopeptide Spot. The composition and chromatographic behavior indicate that GP-2 is identical to the glycopeptide GP-ia isolated by 55 Figure 3. Chromatography of M, Mygodeikticus glyco- peptide GP-2 on a 2.5 x 75 cm column of BioRad Aminex AG-50W-X2 resin. The column was eluted with 200 ml 0.2 M pH 2.9 pyridine-formate buffer and 1000 ml of a gradient from 0.2 M pH 2.9 to 1.0 M pH 5.3 Pyridine-formate buffer. The column eluate was collected in 5 ml fractions and analyzed by evaporating selected tubes to dryness, adding 5 ml water, and reading the absorbance at 240 mu. 56 CHROMATOGRAPHY OF M. lysodeikticus GLYCOPEPTIDE GP-2 7.0 '— 5.0-- .‘t c: I ABSORBANCE -,_- w b I I °-°""- I . I I I Is 20 25 '30 FRACTION NUMBER Figure 3 57 TABLE IV Amino Acid Analysis of M. lysodeiktggus Cell Wall Saccharide A-1 and Glycopeptides GP-i and GP-2 All values are eXpressed in umoles: muramic acid and glucosamine values are corrected for losses incurred during 24 hr hydrolysis. The analyses did not include basic amino acids, so lysine is absent. Muramic Glutamic Fraction Acid Glucosamine ,Acid Glycine Alanine A-i 2.9 2.8 0.095 0.099 0.13 GP-i 1.5 1.6 1.5 1.5 3.1 GP-2 1.5 1.6 0.78 0.75 1.6 58 by Mirelman and Sharon.°9 Purification of N-Acetyl Glucosamine Tetramer A 100 mg sample of the tetramer of N-acetyl glucosamine, originally prepared by charcoal-celite column fractionation of a chitin hydrolysate (prepared by G. Stone), was dissolved in a 2 m1 redistilled water and placed on the Sephadex G-15 column of the previously described automated gel filtration apparatus. The column was eluted with boiled redistilled water at a rate of 10 ml/hr: the column eluate was monitored at 230 mu and collected in 3 ml fractions. The A230 trace displayed one large symetrical peak at an elution volume of 225 ml: the tetramer is therefore quite similar to M, lysodeikticus tetrasaccharide in that they display very similar elution characteristics on the Sephadex G-15 column. Two small trailing peaks were observed: they were attributed to lower molecular weight impurities. Fractions containing the major peak were pooled and lyophilized to produce 66 mg of N-acetyl glucosamine tetramer. Purification of N-Acetyl Glucosamine Hexamer A sample (100 mg) of N-acetyl glucosamine hexamer originally prepared by charcoal-celite column chromatography of a chitin hydrolysate (prepared by G. Stone) was dissolved in 2 ml redistilled water and placed on the Sephadex G-15 column of the automated gel filtration apparatus, and eluted 59 with boiled redistilled water at a rate of 10 ml/hr. The column eluate was monitored at 230 mu and collected in 5 ml fractions. The A230 trace formed one major peak at 300 ml elution volume: The chromatographic behavior of this N-acetyl glucosamine polymer was therefore very similar to that displayed by Fraction 1, which contained M, lysodeik- .El222 cell wall hexasaccharide. Two minor peaks indicating contamination of the original sample were also present. Fractions containing the major peak were pooled and lyophil- ized to yield 38 mg of purified hexamer. Preparation of N-Ethyl Maleimide Inactivated LysozyMe Worthington lysozyme (100 mg) and dithiothreitol (200 mg) were dissolved in 10 ml of 6 M guanidine-hydrochloride which was 0.06 M in pH 8.2 phOSphate buffer. The pH of the resulting solution was adjusted back to 8.2 with 2 N sodium hydroxide solution. The solution was gassed with nitrogen to remove air: the container was then stoppered to exclude air and kept in the dark at room temperature for 6 hours. N-ethyl malemide (440 mg) was added: the pH of the resulting solution was quickly adjusted to pH 6.8 with 2 N hydrochloric acid. The reaction mixture was held at room temperature in the dark for 20 min, then placed in 16/32" sausage casing and dialyzed against two 2 liter volumes of distilled water. The resulting precipitate was washed twice with two 10 ml portions of redistilled water and lyophilized to yield 72 mg of amorphous white powder. 60 The activity of this N-ethyl maleimide modified lyso- zyme was checked by suSpending 1 mg of the modified lysozyme in 3 ml M, lysodeikticus cell suspension (13 mg M, lysodeik- Eiggg cells suspended in a mixture of 90 ml 0.066 M pH 6.5 phosphate buffer and 10 ml 1.0 M sodium chloride solution). The turbidity of the'suSpension as measured by absorbance at 550 mu (Beckman Model B spectrophotometer) did not change over a 2 hour period (A550 remained constant at 0.62): hence this modified lysozyme was completely inactive.‘ Preparation of Reduced, CarboxyMethylated LysozyMe Reduced, carboxymethylated lysozyme was prepared by a modification of the method described by Crestfieild, Stein and Moore.70 Lysozyme (Worthington, 50 mg) was dissolved in 12 ml of an 8 M urea solution prepared by mixing deionized urea (5.78 g), EDTA (24 mg), and pH 8.6 Tris buffer (5.3 g Tris and 9 ml 1.0 N HCl made up to 30 ml with redistilled water) with sufficient distilled water to make a final volume of 12 ml. After 20 min at room temperature, 200 mg of dithio- threitol was added: the resulting solution was kept in the dark at room temperature for 4 hr. A solution of 630 mg of iodcacetic acid in 3.4 ml 0.95 M sodium hydroxide solution was then added: after 20 minutes reaction in the dark at room temperature, the solution was placed in 16/32" sausage casing and dialyzed against 5 changes of redistilled water in the dark. The resulting white precipitate was washed 61 three times in 10 ml portions of redistilled water by suspen- sion followed by centrifugation (clinical centrifuge) and then lyophilized to produce 35 mg of white, amorphous powder. Amino acid analysis of a hydrolysate (5 mg, constant boiling HCl, 105°, 24 hr) of this modified lysozyme indicated that no cysteine or cystine was present. Enzymatic activity of the reduced, carboxymethylated lysozyme preparation, as measured by turbidity (A550) changes in a M,;ysodeikticus cell suspension (1 mg of modified lysozyme added to 3 ml of a suspension of 15 mg cells in a mixture of 90 ml 0.06 M pH 6.5 phosphate buffer and 10 ml 1.0 M sodium chloride solution) was undetectable. Detection of Muramic Acid and Glucosamine in Lysozyme- Substrate Reaction Mixtures The hydrolysis mixtures of the substrates utilized in lysozyme-substrate reactions contain muramic acid and gluco- samine: since these compounds yield discrete peaks on the amino acid analyzer, their presence in hydrolysates of lysozyme-substrate reaction mixtures could be quantitatively determined. Since both muramic acid and glucosamine were exten- sively degraded during the hydrolysis process, a correction factor for this loss was determined by hydrolyzing (2 ml constant boiling HCl, 105°, 24 hr) 1 umole amounts of muramic acid and glucosamine and subjecting the resulting hydrolysate to amino acid analysis. The corrected amino acid analyzer 62 constants for muramic acid and glucosamine were respectively 9.43 and 16.1 area units/mg (normal amino acid constants lie in a range between 24 and 26 area units/mg). Application of this correction factor allowed a quan- titative determination of N-acetyl muramic acid and N-acetyl glucosamine as present in the lysozyme-substrate reaction mixture prior to hydrolysis. These values in turn allowed a determination of the amount of substrate present in the lysozyme-substrate reaction mixtures. In each case, the amount of substrate present was expressed as the umolar ratio of glucosamine to phenylala- nine: since hen's egg white lysozyme contains 3 phenylala- nine residues, a ratio of 0.33 (1 umole of glucosamine/3 umoles phenylalanine) indicated that one N-acetyl glucosamine residue per mole of lysozyme was present. Non-Aqueous Lysozyme-Substrate Reactions: Dimethyl Sulfoxide Quenching Worthington lysozyme (10 mg) and substrate (5 mg of M. lysodeikticus "Fraction 1" hexasaccharide-glycopeptide mixture or tetrasaccharide) were suspended in 1 ml anhydrous glycerol and stored at room temperature (23°) in a dessica- tor over phosphorus pentaoxide for a determined incubation time. The reaction mixture was then quenched with dimethyl sulfoxide (3 ml), held at room temperature for 2 hr, and dialyzed against 6 changes (50 m1) of 8 M urea solution (the 63 final 2 changes were 0.1 M, pH 8.2 in phosphate buffer), or against 6 changes of 6 M guanidine hydrochloride solution (in which case the dialysis bag liquid was made up to 0.1 M pH 8.2 phosphate by the addition of a solid buffer system). Dithiothreitol (100-200 mg) was added to the pH 8.2 dialysis bag liquid, and the solution pH adjusted back to 8.2 by addition of 2 N sodium hydroxide solution. After 4 hr at room temperature, 200-600 mg of N-ethyl maleimide was added. This resolution was dialyzed (16/32" sausage casing) against 6 changes (50 ml) of 8 M urea or 6 M guanidine hydrochloride after 15 minutes reaction time. Dialysis was continued against 6 changes of redistilled water. The resulting precipitate was centrifuged and washed 10 times with 1 ml portions of redistilled water, lyophilized, hydro- 1yzed (2 m1 constant boiling HCl, 105°, 24 hr) and subjected to amino acid analysis (Table XIX). Blank runs consisted of a suspension of reduced, N-ethyl-maleimide-treated lysozyme or reduced, iodcacetate- treated lysozyme (10 mg) and substrate (5 mg)iJ11nfl.of anhy- drous glycerol. This suspension was subjected to the dialysis- washing procedure described in the "live run" experiment, but a change of the dialysis sac was substituted for the inactivation step (Tables XX and XXI). Non-Aqueous_Ly§25yme-Subst;ate Reactions Utilizing_Urea Quenching Lysozyme (Worthington, 10 mg) and M, lysodeikticus 64 cell wall Fraction 1 (mixture of hexasaccharide and glyco- peptide, 5 mg) were suSpended in 1 ml anhydrous glycerol and stored in a desicator over phosphorus pentoxide for the stated incubation time. The reaction mixtures were quenched by the addition of 100 mg of solid urea and 2 ml of 8 M urea solu- tion. The resulting solutions were treated by one of two procedures: (a) dialysis (16/32" sausage casing) against pH 8.2 8 M urea solution (0.25 M in sodium bicarbonate) or (b) dialysis against 6 changes of unbuffered 8 M urea solu- tion, in which case the dialysis sac liquid was lyophilized and dissolved in pH 8.2 8 M urea (0.17 M phOSphate buffer) to bring it to pH 8.2. Dithiothreitol (200 mg) was added to the pH 8.2 dia- lysis sac liquid in both cases: after 5 hours at room tem- perature, a solution of 630 mg iodcacetic acid in 3.4 ml 1 N sodium hydroxide solution was added, When 15 minutes had elapsed, the reaction mixture was successively dialyzed against 6 changes of 8 M urea and 3 changes of distilled water. The precipitates were centrifuged and washed 10 times (clinical centrifuge), and then hydrolyzed (2 ml constant boiling HCl, 105°, 24 hr) and subjected to amino acid analy- sis (Table XXII). Blank runs were prepared by two procedures: (a) Reduced, carboxymethylated lysozyme (10 mg) and Fraction 1 (5 mg) were suSpended in 1 ml anhydrous glycerol. When the required reaction period had elapsed, 100 mg of solid urea 65 and 2 ml 8 M urea solution were added to the glycerol solu- tion. This solution was treated as detailed in the descrip- tion of the "live" run experiments with the exception of the iodcacetate treatment: this was replaced by a dialysis sac change (Table XXIID: (b) reduced, carboxymethylated (RCM-) lysozyme or reduced, N-ethyl maleimide treated (RNEM~) lyso- zyme (10 mg) and M, Mysodeikticus cell wall Fraction 1 were dissolved in 1 ml 6 M guanidine hydrochloride or 8 M urea solution. This solution was dialyzed (16/32 sausage casing) against 12 changes (50 ml) of 6 M guanidine hydrochloride or 8 M urea solution and 6 changes (50 ml) of redistilled water. The resulting precipitate was centrifuged (clinical centri- fuge) and washed 10 times with 1 ml portions of redistilled water. The washed precipitate was lyophilized, hydrolyzed (2 ml constant boiling HCl, 105°, 24 hr), and analyzed for amino acid content (Table XXIV). Aqueous Lygggyme-Substrate Reactions Lysozyme (Worthington, 10 mg) and substrate (5 mg of M, lysodeikticus Fraction 1 hexasaccharide-glycopeptide mix- ture, hexasaccharide, glycopeptide GP-l, glycopeptide GP-2 or tetrasaccharide, or N-acetyl glucosamine hexamer or tetramer) were dissolved.in 1 ml pH 8.2 0.1 M phosphate buf- fer. The resulting reaction mixture was incubated at room temperature (23°) for 15 minutes: then 1.5 g granular 66 guanidine hydrochloride was added to stop the reaction. After 30 minutes at room temperature, the pH of the quenched reaction mixture was adjusted to 8.2 by addition of 2 N sodium hydroxide solution. Dithiothreitol (100 mg) was added and the solution pH was again adjusted to 8.2. The reduction mixture was allowed to stand at room temperature in the dark for 5 hr. The pH was then adjusted to 6.8 with 2 N hydrochloric acid and 220 mg of N-ethyl maleimide was added. After 15 minutes reaction at room temperature in the dark, the solution was placed in a 16/32" sausage casing bag and was dialyzed against two 2-1 changes of distilled water. The resulting precipitate was treated by one of two methods: (a) The precipitate was dissolved in 6 m1 6 M guanidine hydrochloride solution and dialyzed against two 2-1 changes of distilled water. This step was repeated three times. The precipitate was centrifuged and washed ten times with 2 m1 portions of redistilled water, hydrolyzed (2 ml constant boiling HCl, 105°, 24 hr), and placed on the amino acid analyzer (Table XXV). (b) The precipitate was repre- cipitated three times as in (a) and lyophilized. The lyophil- ized material was dissolved in 2 m1 5S formic acid. The formic acid solution was placed on a 20 x 1 cm Biogel P-4 column and eluted with 5% formic acid at a rate of 25 ml/hr. The eluate was collected in 3 ml fractions and analyzed by measuring the.A280 of each fraction. A single peak at 9 m1 elution volume was observed. The peak fractions were lyophilized to yield 3 mg of white solid which was 67 hydrolyzed (2 m1 constant boiling HCl, 105°, 24 hr). and placed on the amino acid analyzer (Table XXV). Blanks were prepared by dissolving 10 mg of reduced, carboxymethylated lysozyme or reduced; Néethyl maleimide treated lysozyme and 5 mg of substrate in 1 ml 6 M guanidine hydrochloride solution. This solution was dialyzed (16/32 sausage casing bag) against two 2-1 changes of distilled water. The resulting precipitate was then treated by either method (a) or (b) as previously described (Table XXVI). Several glycopeptide GP-2 blank preparations utilized gel filtration exclusively. Glycopeptide GP-Z (5 mg) and 10 mg of either reduced, carboxymethylated lysozyme or reduced, N-ethyl maleimide treated lysozyme were dissolved in 1 ml 6 M guanidine hydrochloride and dialyzed (16/32 sausage casing bag) against two 2-1 changes of distilled water. The result— ing precipitate was centrifuged, washed twice with 2 ml por- tions of redistilled water, and dissolved in 2 ml 5% formic acid. This solution was chromatographed on a 1 x 20 cm BioGel P-4 column with 5% formic acid at an elution rate of 25 ml/hr. The A280 peak from this column was lyophilized and rechromatographed on the same P-4 column with 5% formic acid. The A280 peak was lyophilized and hydrolyzed (2 ml constant boiling HCl, 105°, 24 hr), and then subjected to amino acid analysis (Table XXVI). 68 Effect of Hydroxylamine on the Mysozyme-Glycopeptide GP-2 Reaction Lysozyme (Worthington, 20 mg) and glycopeptide GP-2 (10 mg) were dissolved in 2.0 ml pH 8.2 0.1 M phosphate buffer and allowed to stand at room temperature (23°) for 15 minutes. The reaction was quenched by adding 3.0 g of solid guanidine hydrochloride. The pH of this solution was adjusted to 8.3 with 2 N sodium hydroxide solution and 400 mg of dithiothreitol was added. After adjusting to pH 8.3, the reaction mixture was kept under nitrogen atmosphere in the dark for 5 hr. N-ethyl maleimide (840 mg) was added and the solution pH adjusted to 6.8 with 2 N hydrochloric acid. When 15 minutes had elapsed, the reaction mixture was dialyzed (16/32" sausage casing bag) against two 2-1 changes of dis- tilled water. The resulting precipitate was centrifuged, washed twice, and then lyophilized. The lyophilized white solid was dissolved in 3 ml 5% formic acid, placed on a 20 x 1 cm BioGel P-4 column, and eluted with 5% formic acid at a rate of 25 ml/hr. The eluate A280 peak fractions were lyophilized and rechromatographed on the same P-4 column. The eluate A280 peak was lyophilized and divided into two portions. The first portion was hydrolyzed (constant boiling HCl, 105°, 24 hr) and analyzed for amino acid content (Table XXVII). The remaining portion of lyophilized material was dis- solved in 2 ml'6 M guanidine hydrochloride solution 1.0 M in 69 hydroxylamine and 0.06 M in phosphate buffer. This solution was kept at room temperature for 12 hr and then dialyzed (16/32 sausage casing bag) against two 2-1 changes of dis- tilled water. The precipitate was centrifuged and washed twice with 2 ml portions of distilled water. The washed precipitate was dissolved in 2 ml 5% formic acid and chromatographed on a 20 x 1 cm BioGel P-4 column with 5% formic acid at an elu- tion rate of 25 ml/hr. The eluate A280 peak was lyophilized, hydrolyzed (constant boiling HCl, 105°, 24 hr) and subjected to amino acid analysis (Table XXVII). RESULTS TABLE VI Hydrolysis Rate of 2-Methzlthioacetaldehyde Diethl Acetal in Dioxane-Water The reaction mixture consisted of 10.8 to 14.4 mg 2-methylthioacetaldehyde diethyl acetal, 5 m1 of dioxane, and 5 ml of perchloric acid. Absorbance was measured at 275 mu in a 1-cm cell. Absorption increment time intervals ranged from 150-300 seconds depending on the hydrolysis rate. Perchloric Acid, M P 10“ k1 102 k2 0.005 0.010 1.22 2.44 0.010 0.020 2.19 2.19 0.020 0.040 4.93 2.46 0.020 0.040 4.93 2.46 0.025 0.050 5.21 2.10 0.025 0.050 5.10 2.05 Average = 2.28 70 71 TABLE VII Mydrolysis Rate of 2-MethoxzacetaldeMyde Diethyl Acetal in Dioxane-Water The reaction mixture contained 45 to 99 m8 2-methoxy- acetaldehyde diethyl acetal, 10 ml of dioxane and 10 ml of perchloric acid. Absorbance was measured at 270 mu in a 10-cm cell. Absorption increment time intervals ranges from 100-300 seconds, depending on hydrolysis rate. Hammet acidity values (Ho) were obtained from data cited by Kreevoy and Taft.72 Perchloric 4 4 Log k1 Acid, M Ho 10 k1 10 k2 + H0 Log k2 0.5u3 0.10 “.90 9.0“ -3021 -300” 005123 0010 ”.80 8085 -3022 -3005 00990 -001'3 21.2 21.6 -2080 -2061 The value of k obtained by extrapolation of Lag k g0 seg'1 (k /HC1015, cone) to ze ional strength was 2.63 x 10' 1 '1 ( igure 4). 72 TABLE VIII Mydrolysis Rate of Z-Ethoxyacetaldehyde Diethyl Acetal in Dioxane-Water The reaction mixture contained 27 to 97 m8 2-ethoxy- acetaldehyde diethyl acetal, 10 ml of dioxane, and 10 ml of perchloric acid. Absorbance was measured at 270 mu in a 10- cm cell. Absorption increment time intervals ranged from 100-300 seconds. Perchloric 4 4 Log k 1 Acid, M Ho 10 k1 10 k2 + H0 Log k2 00290 0.22 1.41 “.90 -3063 -3031 00290 0.22 1.91 6070 -3050 -3017 00543 0.10 6.92 12.8 '3010 -2081 00543 0010 7005 1300 “3010 -2081 0.990 -0.13 62.0 62.7 -3.34 -2.20 0.990 -0.13 35.1 35.5 -3.59 -3.45 The value of k obtained by exfirapolation of Log k2 to zero ional strengtfi was 1.70 x 10’ sec' mole“ (Figure 4). 73 .mcoaposdahopoo mo pom some how House Hopnoaaaoawo on» opooaosd mosda Hooapnob was .soapoapsoosoo odes odHoHSoaom once on oopoaoaonpxo one: Hmpooo ahspoao oohsooaopooo Acllllsv :hNOSpoIN one ANIIIINV IhHoSpoaIN mo moves mamhaoaohs one .Hmpood flagpoam thNospmnm one Ihnospoznm mo mopom mamhaoaohm mo :oapoaoaonpwm .: ohawam 74 0g ad Nd #0 09w: V 0530mm no no so no so .3 III oullnll. .Illllxrlllnlull III-III! 1 'I I ' 4P .ooN: 75 TABLE IX Mydrolysis Rate of 4-Methzlthiobutraldehzde DietMyl Acetal in Dioxane-Water A stock solution of 39.2 mg 4—methylthiobutraldehyde diethyl acetal in 15 m1 of dioxane was prepared: 5 ml por- tions of this solution were mixed with 5 ml of perchloric acid to form the reaction mixture. Absorbance was measured at 270 mu in a 1-cm cell. The absorption increment time interval was 300 seconds. Perchloric Acid, M F 103 k1 101 k2 0.010 0.020 ' 1.30 1.30 0.010 0.020 1.30 1.30 Average = 1.30 76 TABLE X Hydrolysis Rate of Valeraldehyge Diethyl Acetal in Dioxane-Water Two stock solutions were prepared containing 36 and 83 mg valeraldehyde diethyl acetal per 15 ml of dioxane: 5 m1 portions of these solutions were mixed with 5 ml of per- chloric acid to form the reaction mixture. Absorbance was measured at 270 mu in a i-cm cell. The absorbance incre- ment time interval was 300 seconds. __A4.k 1 Perchloric Acid, M P 103 k1 10 k2 0.010 0.020 1.85 1.85 0.010 0.020 1.87 1.87 0.010 0.020 1.81 1.81 Average = 1.84 77 TABLE XI Hydrolysis Rate of 5-Methoxy-24Pentanone Diethyl Ketal in Dioxane-Water Stock solutions containing 174 to 177 mg of 5-methoxy- Z-pentanone diethyl ketal per 20 m1 of dioxane were prepared: 5 ml portions of these solutions were mixed with 5 ml of acetate buffer to form the reaction mixture. Absorbance was measured at 270 mu in a 1-cm cell. The absorption increment time interval ranged from 300-600 seconds depending on the rate of hydrolysis. The notation (corr.) indicates that the hydrolysis rate was corrected for secondary salt effects. Acetic 4 10'?2 k Acid, M Acetate, M r 10 k1 (corr.3 10"2 k2 0.00375 0.00125 0.0025 4.34 3.65 4.77 0.00375 0.00125 0.0025 4.26 3.57 4.66 0.0250 0.0125 0.025 3.69 3.58 7.02 0.0250 0.0125 0.025 3.68 3.57 7.00 0.0375 0.0125 0.050 6.58 3.41 8.34 0.0375 0.0125 0.050 6.83 3.54 8.77 0.0375 0.0125 0.100 9.66 3.97 12.25 0.0375 0.0125 0.100 9.48 3.91 12.05 value of it”2 (Figure 5). value of 10'2 k k2 extrapolated to zero ional strength 2 4.20 (corr.) extrapolated to zero ional strength = 3.55 (Figure E). ‘ 78 .msoapodaahopoo go now some you Moshe Hopnoadhomwo on» opooaosa modda Hooapaob one .Spwsoapm Hosoa once on oopoaoaoapwo ones money mammaoaoms Hopox thpoao cacao» InoalmnmNoSpoaum A.IIII.V oopooaaoonpoommo paomlhaoocooom can A III: v oopoonuoocs Spom .opwm mamhaoadhm Hopom thpoan oaosmusomlmlhxoSQmZIm mo nodeHoaoame .m oaswam 79 n 0.3m: IhOmehm 44.20. 9.0 cod mod nod cod no.0 V0.0 no.0 No.0 5.0 II \ .I 00.0 I. a _ _ _ a _ _ . _ _ loos db F..\\..1 I 03. 7y I . I com 7.... I. \ , l 8.2. I...\ (oom— _ _ _ _ _ a r _ _ L. 3593129295: :EEE mzozfizmadlrxofmzn do zozfioaéaxa 80 TABLE XII Mydrolysis Rate of 5-Methylthio-2-Pentanone Diethyl Ketal in Dioxane-Water Stock solutions containing 180 to 188 mg of 5—methyl- thio-2-pentanone diethyl ketal in 20 ml of dioxane were pre- pared: the reaction mixture contained 5 ml of this stock solution and 5 ml of acetate buffer. Absorbance was measured at 275 mu in a 1-cm cell. The absorbance increment time interval was 600 seconds. The notation (corr.) denotes hydrolysis values which have been corrected for primary salt effects. . figigtcn .Acetate, M i P 10” k1 (gorr43 10"2 k2 0.00435 0.00125 0.0025 3.06 2.57 3.36 0.00435 0.00125 0.0025 3.10 2.60 3.40 0.025 0.0125 0.025 2.77 2.67 5.26 0.025 0.0125 0.025 2.77 2.67 5.26 0.025 0.0125 0.025 2.67 2.59 5.08 0.0025 0.0025 0.050 1.71 2.67 6.75 0.0025 0.0025 0.050 1.95 3.03 7.42 0.00375 0.00125 0.050 5.74 3.00 7.29 0.0125 0.0125 0.050 1.91 2.96 7.28 0.0125 0.0125 0.050 1.92 2.98 7.32 0.0188 0.0065 0.050 5.72 2.99 7.26 0.025 0.025 0.050 1.89 2.94 7.19 81 TABLE XII (Continued) .? Agigfou Acetate, M I" 104k1 (go;r¥3 10"2 k2 0.025 0.025 0.050 1.94 3.01 7.38 0.0375 0.0125 0.050 5.77 3.01 7.32 0.0758 0.0372 0.0744 3.81 3.18 7.25 0.0758 0.0372 0.0744 3.79 3.16 7.21 0.0345 0.0125 0.100 7.62 3.14 9.67 0.0345 0.0125 0.100 7.67 3.16 9.72 Value of 10"2 k (Figure 6). value of 10"2 k 2 extrapolated to zero ional strength 3 3.16 (corr.) extrapolated to zero ional strength = 2.60 (Figure 8). 82 .mcoapocaaaopoo no pom some now house Hopsoaaaoawo on» cascaded moaaa aooapaob one .Spwsoapm Homo“ open on dopoaoaoapwo one: nouns mamhaonoh: Hdpox thpoao ozozspsoa INIodanmSpoaIm A IIII V oopooaaooIpooumo pHdthnmosooom use AIIIIIIV oopooaaoosd Spom .opmm mamhaoaohm HopoM Hanpoan oeossasoarmaoaseaascozum co soapsaoadspwm .o caewaa 83 o oaau_m IhOZmahm ._._0~.n_>z ._I._.m_o mZOZI.—m<v mow/10mm; ssnow .l. CON _ a _ a _ _ _ ._ImodIhm<40~_O>I >m mmzmon<$uzmzuu 1.0 29503»: a: z. 8.32; mafia”. ._oz<_.:.m 94 .oaspwda namhaonohs on» no madmaon dososoad no hands an oosaahopod no: ososdpsoAImlodSthnpoaIm maduhaohdh: an encodes Hosanna .Hopom thpoan ocosspaom Imuoazaaasccznm weanaaoaeam as oncoaom Hosanna .a osswam 95 0 oa:m_m 'IVE)! 310W\10NVHI3 SQ‘IOW' 3283. Eva ._ in s 88 - 88 coo. _ _ q _ _ . _ I Iol II 163 I‘ll _ 4I._.m<._O¢Q>I >0 mm- .. 083scnzc|zn CH2H+ 25 002115 002H5 CH I 3 so 0 / \ fast 5 01124le i 033-8-082- -H + 02115011 0C2H5 Enzymatic assays of 2-methy1thioacetaldehyde diethyl acetal hydrolysis mixtures indicate that ethanol is released in a manner consistant with first order kinetics (Figure 7-8). No initial rapid release of ethanol is noted. This indicates that if an intermediate cyclic sulfonium ion is present dur- ing the hydrolysis of this acetal, its formation is the rate determining step of the reaction. If cyclic sulfonium ion decomposition were rate determining, a rapid release of one mole of ethanol per mole of acetal concomitant with the rapid formation of the ion would be apparent. The hydrolysis rate of 4umethylthiobutraldehyde diethyl acetal differs very little from that of the parent acetal, valeraldehyde diethyl acetal: 108 0C H |25 IX CHB-S-CHZCHZCHZ-C-H 0°2H5 4~Methy1thiobutra1dehyde Diethyl Acetal 1 1 k2 = 0.130 sec" mole- 0C2H5 X CHB-CHZCHZCHZ-C-H OCZH5 Valeraldehyde Diethyl Acetal k2 = 0.184 sec"1 mole-1 In this case inductive effects are negated by the distance of the inductive group from the reactive site. Apparently no anchimeric assistance is operative in this case even though the formation of a 6-membered cyclic sul- fonium ion would appear to be more favorable than the for- mation of a 3-membered cyclic sulfonium ion. Ketal Mydrolysis rates The hydrolysis rates of the ketals of 5-methoxy-2- pentanone and 5-methylthio-2-pentanone exhibit a small salt effect even when the second-order hydrolysis rates are cor- rected for secondary salt effects (Figures 4 and 5): there- fore the hydrolysis rates of these ketals were extrapolated to zero ional strength. The magnitude of this residual salt effect is so small that no mechanistic implications may be attached to it. 109 The corrected hydrolysis rates of the substituted ketals are slightly slower than that of the parent ketal: $C2H5 XI CHBSCHZCHZCHZ-C'i-CH3 OCZH5 5-Methylthio-24Pentanone Diethyl Ketal k2 = 3.55 x 10""2 sec-1 mole"1 002B5 XII CHBOCHZCHZCHz-C-CH3 OCZHS 5-Methoxy-24Pentanone Diethyl Ketal -2 -1 1 k2 = 2.60 x 10 sec mole- OCZHS XIII CH3CH20H2-l-CH3 OCZHS ZePentanone Diethyl Ketal -1 1 k2 = 8.59 x 10.2 sec mole- Again, the disparity in hydrolysis rate is small when the substituted ketals are compared with the parent ketal and can be accounted for by small magnitude inductive and field effects. The rate of evolution of ethanol from 5- methylthio-Z-pentanone diethyl ketal hydrolysis reaction mixtures is consistant with first-order kinetics and does not indicate the formation of a cyclic sulfonium ion (Figure 9). 110 Hydrolysis of tetraMydropygan ethers The 2-alkoxy substituted tetrahydropyran system bears a marked resemblance to structures commonly associated with glycosides; As such, the alkoxy-tetrahydropyrans would appear to be ideal model compounds in the study of glycoside hydrolysis since they allow direct observation of 01-0 bond hydrolysis free from aberraticns created by adjacent hydroxyl groups. These ethers are therefore ideally suited for a demonstration of anchimeric assistance in glycoside model hydrolysis. Unfortunately, the hydrolysis rates of all the alkoxy- substituted tetrahydropyrans which were investigated were quite similar: 2-Ethoxytetrahydropyran k = 1.53 x 10'2 sec"1 mole- 2 <::>.0-cnzcnz-s-CH3 2-(Z-Methylthioethoxy)-Tetrahydropyran k2 = 1.61 x 10"2 sec"1 mole"1 1 XV 111 < >..0-CHZCH2-0-CH3 XVI ' 2-(2-Methoxyethoxy)~Tetrahydropyran k = 2.05 x 10'"2 sec": mole'l 2 1.73 x 10' sec' mole"1 (XVIII) . N— 0-CH CH HQ XVII 2-(2-Pyridinylethoxy)-Tetrahydropyran k2 = 1.91 x 10"2 sec-1 mole-1 0.00 in acetate buffer No evidence for anchimeric assistance is noted in the case of the hydrolysis of the tetrahydropyran ethers: XIV, XV, and XVI exhibit remarkably similar hydrolysis rates in dioxane-water. The hydrolysis rates of XVI and XVII in water as determined by hydrogen peroxide assay also are very similar. The latter results indicate that protonated hetero- cyclic nitrogen does not participate in anchimeric assistance. Since XVII did not hydrolyze at a measurable rate in acetate buffer of pH 4.64, a pH at which the pyridine nitrogen is only partially protonated, the possibility that unprotonated heterocylcic nitrogen participates in anchimeric assistance is also remote. The lack of evidence for anchimeric assistance by a nitrogen-containing heterocycle is disappointing since pre- vious experiments in this laboratory indicated that the 112 following imidazole-substituted ketal is capable of anchimeric 76 $0283 £[::]l-0H2CH2-T-CHB OCZH3 4(5)-(y-Oxobutyl)-Imidazole Diethyl Ketal assistance: It should be noted that repeated attempts to synthe— size a imidazole-substituted tetrahydropyran failed to yield a nitrogen-containing product. It is apparent that the only probable instance of anchimeric assistance in the hydrolysis rate of the model glycosides discussed is that which occurs during the hydro- lysis of 2-methylthioacetaldehyde diethyl acetal. This demonstration of anchimeric assistance by the sulfur atom of the methylthio group becomes more important when it is related to glycosidase catalysis. It is entirely possible that the methionine methylthio group participates in glyco- sidase catalysis with consequent formation of a covalent glycosyl-sulfonium ion-enzyme intermediate. LysozyMe-Substrate Reactions Non-ggueous reactions Non-aqueous lysozyme-substrate reactions were carried out in nearly anhydrous glycerol (the small amount of water contributed by the water of hydration of lysozyme and sub- strate was impossible to exclude). Glycerol was chosen as a 113 solvent for several reasons: (a) glycerol does not appreci- ably perturb the native configuration of lysozyme,77 (b) both lysozyme and substrate are slowly soluble in glycerol, and (c) lysozyme retains its enzymatic activity in glycerol as demonstrated by its ability to slowly solubilize M, My§odeikticus cell walls suspended in glycerol. The very low concentration of water in the lysozyme- substrate reactions conducted in glycerol should decelerate the reaction between enzyme and substrate and allow a demon- stration of the existence of a stable enzyme-substrate intermediate if one indeed exists. Quenching of glycerol-solvated lysozyme-substrate reactions with dimethyl sulfoxide, followed by exhaustive washing procedures, produced positive results in the case of M, lysodeikticus Fr. 1 hexasaccharide-glycopeptide mixture (Table XIX). Hydrolysates of lysozyme Fr. 1 reaction mix- tures yielded sufficient glucosamine and muramic acid to give glucosamine/phenylalanine ratios which ranged up to 0.30 (approximately one mole of glucosamine per mole of lysozyme). Hydrolysates of lysozyme-M, Mygodeikticus tetrasac- charide prepared under the same conditions yielded no detec- table muramic acid or glucosamine. This set of experiments also indicated that 6 M guanidine hydrochloride solution is a much more effective denaturing agent and dialyzing agent than 8 M urea solution. The secondary and tertiary structure of lysozyme is completely disrupted by even 5 M guanidine 114 78 hydrochloride solutions: this is not the case in urea solu- tions, even at 8 M concentrations.79 Indeed, lysozyme dis- plays a low level of lytic activity in 8 M urea as measured by lysis of bacteria80 and solubilization of M, lysodeikticus cell walls.81 This behavior probably accounts for the low level of binding observed when 8 M urea solutions were utilized as a dialysis medium: the lysozyme may have recovered some activity subsequent to quenching and prior to reduction and alkylation. In contrast to the binding observed in "live" runs, glycerol-solvated blank runs utilizing reduced, N-ethyl- maleimide-treated lysozyme and M, Mygodeikticus Fr. 1 give no evidence for binding (Table XX) when the N-ethylmaleimide- treated lysozyme is completely inactive: the low level of binding observed in some of the blank runs can be attributed to the low level of lytic activity retained by these prepara- tions (after this incident, the lytic activity of all blank lysozyme preparations was carefully checked). Glycerol-solvated blank runs utilizing reduced, car- boxymethylated lysozyme and M, lysodeikticus Fr. 1 also give no evidence for binding (Table XXI). 0n the other hand, glycerol-solvated lysozyme-substrate reactions utilizing urea quenching gave equivocal results. "Live" runs utilizing urea quenching and carboxymethylation resulted in reaction mixture hydrolysates which contained substantial amounts of muramic acid and glucosamine (Table XXII). However, blank runs utilizing either reduced, 115 carboxymethyl lysozyme or reduced N-ethylmaleimide treated lysozyme had glucosamine/phenylalanine ratios of the same magnitude (Table XXIII). Lysozyme-substrate blanks prepared without glycerol also gave hydrolysates containing substantial amounts of muramic acid and glucosamine (Table XXIV). Much of the difficulty encountered in these urea-quenched glycerol- mediated lysozyme-substrate reactions may have been due to the previously mentioned inadequacies of urea as a lyso- zyme denaturant: quenching will not be effective if it is not rapid and complete. In summary, glycerol-mediated lysozyme-substrate reactions indicate that the M, lysodeikticus Fr. 1 hexa- saccharide-glycopeptide mixture is the only substrate which demonstrates active binding under the conditions employed in these eXperiments. The highest glucosamine/phenylalanine ratio in the runs with active lysozyme was 0.30: even when this value is compared with the highest blank value of 0.16 (this blank is not analogous because it was prepared under conditions which were not identical with the high-labeling runs with active enzyme) a preferential binding of M. lysodeikticus cell wall hydrolysis products is indicated. These results are especially striking when compared with the negative labeling results encountered with M, lysodeik- tucus tetrasaccharide. 116 Agueous lysozyMe-substrate reactions Short-time lysozyme-substrate reactions carried out by quenching, washing and hydrolyzing 15-minute reaction mixtures confirmed and extended the observations carried out in glycerol (Table XXV). N-acetylglucosamine tetramer and hexamer did not bind under aqueous reaction conditions (the observed gluco- samine/phenylalanine ratio of 0.02 observed in several cases is the detection limit of the assay system: lower muramic acid and glucosamine concentrations yield no visible chromatogram peaks). As observed in the glycerol-mediated lysozyme-sub- strate reactions, Fraction 1 from lysozyme hydrolysates of M, lysodeikticus cell walls strongly binds to reduced, alkylated lysozyme even after exhaustive reprecipitation. The phenonomon of Fraction 1-lysozyme binding was clarified when Fraction 1 was separated into its component glycopeptides and hexasaccharide. Purified M, lysodeikticus hexasaccharide does not bind at all under the conditions employed in the aqueous labeling eXperiments (Table XXV). 0n the other hand, both M, lysodeikticus glycopeptide GP-1 and glycopeptide GP-2 appear to bind to lysozyme very strongly, as evidenced by the high glucosamine/phenylalanine ratio (0.15-0.29). Inactivated lysozyme-glycopeptide blanks in some cases also exhibited apparent incorporation of substrate, but the level was always less than half that found in the 117 eXperiments with active lysozyme (Table XXVI). It is now apparent that substrate binding in both non-aqueous and aqueous lysozyme-substrate reactions is observed only when M, lysodeikticus glycopeptide GP-i or GP-2 is utilized as substrate. In accord with the results obtained in the non- aqueous lysozyme-substrate reactions, the blank runs in the aqueous lysozyme-substrate reactions evidence substrate incorporation, (Table XXVII). However, the high level of incorporation demonstrated by M, Mysodeikticus glycopeptides GP-1 and GP-Z indicates that they preferentially bind to native lysozyme under the conditions of these experiments. The interactions between lysozyme and glycopeptides GP-1 and GP-Z are very strong: the complex survives washing, reprecipitation, and gel filtration. The rigorous isolation methods, eSpecially the gel filtrations, indicate that this complex may be covalent. The nature of the binding observed with these glyco- peptides was investigated by treating a lysozyme GP-2 complex of known substrate concentration with a solution of hydroxy- lamine. If the primary binding interaction is a covalent bond between lysozyme and glycopeptide created by the forma- tion of an ester linkage between a substrate carboxyl group and an enzyme hydroxyl group or between a substrate hydroxyl group and an enzyme carboxyl group, then the hydroxylamine should liberate the substrate by cleaving the ester linkage and forming a hydroxamic acid. In this instance, hydroxy- 118 lamine did not appreciably lower lysozyme-glycopeptide GP-2 binding as evidenced by the glucosamine/phenylalanine ratio (Table XXVI). It appears that the primary binding force in the lysozyme GP-2 complex is not a covalent ester linkage. A comparison of the structures of the binding- and non-binding-substrates indicates that the pentapeptide side chains of the glycopeptides are the distinguishing features of the binding substrates (Figure 10). It should be noted that although glycopeptide GP-i binds to lysozyme, it is the lysozyme-hydrolyzed analogue of GP-2. Since all natural lysozyme substrates (bacterial cell walls) contain peptide chains, it is tempting to speculate that these peptide chains play an important part in the bind- ing of substrate to lysozyme during the catalytic process. These peptide chains may play an important part in lysozyme catalysis by enhancing the enzymatically productive substrate orientations. Hydrogen bonding,82 hydrophobic bonding, and ionic bonding may be important in this type of substrate bonding phenomonon, but no definite assessment of their relative contributions may be made. In summary, then, the glycoside model compound experi- ments indicate that stable covalent intermediates, especially those of the sulfonium ion type, may be of importance in glycosidase-transglycosidase catalysis. The lysozyme-sub- strate eXperiments indicate that a stable intermediate may be 119 .mcoapoMoa opdhmeSM Icahuomma ca omufiaaps mopdhpwfldm mahuomhq .0H mhswdm 120 Figure 10. LYSOZYME SUBSTRATES Non r-Binding Saccharides NAc N N-Acefyl 9 la cosomine Tetramer: R=H MJysodeikticg; Tetrasacchoride: R=CH3§HCOOH H20H H20H CH20H H20H e - e e e 0 , . . Ac NAc NAc , CH20H CHZOH cnon ‘ CHZOH CH20H 0'29“ 0 e e e e NAC NAc NAc NAc NAc' N-Acetyl g iucosomine Hexc mer: R=H Me lysodeikticus Hexasaccharide: R=CH3§HCOOH NAc Binding Glchpeptides H2011 CH20H CH20H 01on CH3CHco I;IH l-A la b—V—éu u—a—Gly-COOH D‘fC'Lz lu—c—Gly-COOH L- Lys-e-NH L-Lys-t-NHZ D’é'a N1!) coon co (2) M, brfsodeikticus Glycopepiide GP-i= dashed glycosidic linkage is absent (disocchoride—pentopeptide dimer) fiixsddeikticus Glyc0peptide GP-21pentc1peptide 0 is absent (tetrasaccharide-monopentapeptide) 121 present in the reaction between lysozyme and M, lysodeikticus glycopeptides. The presence of this intermediate is not due to ester formation, and in all probability, it is not due to sulfonium ion formation. 10. 11. 12. 13. 14. 15. 16. 17. BIBLIOGRAPHY Fitderald, E.,and Lapworth, A,, Proc. Chem. 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