LYSOZYME- SUBSTRATE REACTIONS AND HYDROLYSIS 0F GLYCOSIDASE-GLYCOSIDE MODELS Thesis for the Degree of Ph. D; MICHIGAN STATE UNIVERSITY HERMAN NUNEZ 1970 gej.) ( w—v‘ ‘ LIBRARY . M Chigats St‘iei's' ‘8 Univc-zeiiy — 1'13", 'm‘.’ This is to certify that the thesis entitled LYS OZYME-S UBS TELTE RELC TI ON S um HYDROLYSIS OP GLYC OS IDASE-GLYOOSIDE MODELS presented by Hernan Nunez has been accepted towards fulfillment of the requirements for M degree inmaohenistry Major profess r Date ‘zz’l/[r/fl 2— 7/ / $2 0 0-169 .111" ‘4...)- e": W, ' H t It ‘~ 2: ' 8’ magma BY ITEMS 3. SONS’ aunx mm mc. LIBRARY BINDERS “.l a-.— ABSTRACT LYSOZYME-SUBSTRATE REACTIONS AND HYDROLYSIS OF GLYCOSIDASE-GLYCOSIDE MODELS BY Hernan Nunez The catalytic role of the amino acid side chains at the active site of lysozyme was studied. The investigation was carried out with lysozyme itself and with model compounds. The participation of a protein carboxyl or amide group in the lysozyme-catalyzed reaction would result in an acylal or an imidate glycosyl—enzyme intermediate, reSpectively. Among the compounds that possibly would react with these inter— mediates are hydroxylamine, with the acylal, and sodium boro— hydride. with the imidate. Both of these substances were investigated as trapping reagents during the lysozyme- catalyzed reaction. The results indicated that neither of these reagents trapped these intermediates, if present. In the reaction with the model compounds, hydroxylamine formed five percent hydroxamic acid with tetrahydrOpyranyl acetate but sodium borohydride did not reduce methyl or ethyl acetimi- date. I The amino acid side chain involved in a stable and apparently covalent enzyme-substrate complex was also Hernan Nunez investigated. It was found that the marked stability of the enzyme-substrate complex is the result of electrostatic interactions, since high salt concentration, pepsin hydrolysis, and complete enzymatic hydrolysis dissociated the substrate from the enzyme. The following acetals were synthesized, and their hy- drolysis rates in 50% dioxane-water were determined: acetamidoacetaldehyde diethyl acetal, 5-([tetrahydropyran- 2-yl)oxy]-pr0pionic acid methyl ester, 5-[(tetrahydropyran- 2-yl)oxy]-propionic acid, 5-[(tetrahydropyran-Z—yl)oxy]-pro- pionamide, 2-(tetrahydrOpyran-Z-yloxy)-acetic acid ethyl ester, 2-(tetrahydropyran-Z—yloxy)-acetic acid, 2-(tetrahydro- pyran-Z-yloxy)-acetamide, 6-ethoxytetrahydrOpyran-Z-car- boxylic acid ethyl ester, 6-ethoxytetrahydropyran-Z-car- boxylic acid, and 6-ethoxytetrahydr0pyran-2-carboxamide. These glycoside model compounds contain neighboring grOUps which resemble either the amino acid side chains at the lysozyme active site or the acetamido group in some lysozyme substrates. The results indicated that specific acid catalysis gov- erns the hydrolysis of these compounds, i.e., there is no intramolecular catalysis by the acetamido, amide, and carboxyl neighboring group. The effects of the amide, carboxyl and ester substituents in the rate constants for the hydrolysis of the tetrahydro- pyran derivatives indicate that the hydrolysis proceeds via Hernan Nunez a cyclic carbonium ion mechanism. Since the substituent effects on the hydrolysis rate constants of the tetrahydro- pyran derivatives resemble the structural effects on the hydrolysis of glucopyranosides, the results suggest that the glucopyranoside hydrolysis also proceeds via a cyclic car- bonium ion mechanism. LYSOZYME-SUBSTRATE REACTIONS AND HYDROLYSIS OF GLYCOSIDASE-GLYCOSIDE MODELS BY Hernan NunethI A THESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1970 34; V57 6 ~/o“7’° ACKNOWLEDGMENTS The author wishes to express his gratitude and sincere appreciation to Dr. John C. Speck, Jr., for his guidance, support, and constructive criticism throughout the course of this investigation. The author would also like to thank Dr. Gerasimos J. Karabatsos, Dr. Loran Bieber, Dr. William W. Wells, and Dr. Joseph E. Varner for serving on his guid- ance committee and Dr. Richard J. Embs for his valuable assistance in the preparation of this manuscript. The author is eSpecially grateful to his wife, Betty, for her love and encouragement during these studies. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 Approach to the Present Investigation . . . . 1 Structure of the Lysozyme Substrates. . . . . 2 Lysozyme Active Site Structure. . . . . . . . 5 Postulated Reaction MeChanisms. . . . . . 6 Chemical Studies with Glycosidase Model Compounds. . . . . . . . . . . . . . . . . 10 Chemical Studies on the Lysozyme-Catalyzed . Reaction Mechanism . . . . . . . . . . . . 15 Substrate Acetamido Group Participation . . . . 18 EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . 20 Reagents. . . . . . . . . . . . . . . . . . . . 20 General. . . . . . . . . . . . . . . . . . 20 Lysozyme Reagents. . . . . . . . . . . . . 20 Acetal Reagents. . . . . . . . . . . . . . 23 Preparations. . . . . . . . . . . . 24 Acetamidoacetaldehyde Diethyl Acetal . . 24 2- (TetrahydrOpyran-Z-yloxy)-aCetic Acid . Ethyl Ester . . . . . . . . . . . . . 24 Methylhydroxypropionate. . . . . . 25 3- [(TetrahydrOpyran- 2-y1)oxy]-pr0pionic Acid Methyl Ester . . . . . . . . . 26 6- EthoxytetrahydrOpyran-Z-carboxylic Acid Ethyl Ester . . . . . . . . . . . . . 26 Ethyl Acetimidate. . . . . . . . . . . . . 27 Methyl Acetimidate Hydrochloride . . . . . 28 Tetrahydropyranyl Acetate. . . . . . 28 Determination of Rate Constants for Hydrol- ysis of Acetals and Tetrahydropyran Derivatives . . . . . . . . . . . . . 29 Reactions of Hydroxylamine with Tetrahy- drOpyranyl Acetate. . . . . . . . . . 50, Reaction of Hydroxylamine at the Ester Center of Tetrahydropyranyl Acetate . 51 iii TABLE OF CONTENTS-~continued Page Kinetic Measurements of the Spontaneous Hydrolysis and the Reaction of Hydroxylamine at the Acetal Center of Tetrahydropyranyl Acetate . . . . . . 51 Reduction of Ethyl Acetimidate with Sodium Borohydride . . . . . . . . . . . . . 32 Reduction of Methyl Acetimidate Hydro- chloride with Sodium Borohydride. . . 35 Determination of Ethanol Formation in the Sodium Borohydride Reduction of Methyl Acetimidate Hydrochloride. . . 55 Determination of Aldehyde Formation in the Sodium Borohydride Reduction of Imidates. . . . . . . . . . . . . . . 54 Lysozyme Preparations . . . . . . . . . . . . . 54 Preparation of M, luteus Cell Walls. . . . 54 Preparation of M, luteus Glycopeptide GP-2 35 Preparation of Reduced, N-Ethylmaleimide- alkylated Lysozyme. . . . . . . . . . 57 Preparation of Fraction I. . . . . . . . . 38 Preparation of the Enzyme-substrate Complex . . . . . . . . . . . . . . . 59 Detection of Muramic Acid and Glucosamine in the Enzyme-substrate Complexes . . 4O Tryptic Hydrolysis of the Enzyme-substrate Complex . . . . . . . . . . . . . . . 4O Pepsin Hydrolysis of the Enzyme-substrate Complex . . . . . . . . . . . . . . 42 Complete Enzymatic Hydrolysis of the Enzyme- -substrate Complex. . . . . . . 42 Sodium Chloride Effect on the Enzyme- substrate Complex . . . . . . . . . 43 Effect of Sodium Borohydride on the Lysozyme Activity . . . . . . . . . . 44 Effect of Hydroxylamine on the Lysozyme Activity. . . . . . . . . . . . . . . 45 RESULTS. . . . . . . . . . . . . . . . . . . . . . . 46 Lysozyme-substrate Reactions. . . . . . . . . . 48-59 Reduction of Imidates with Sodium Borohydride . . . . . . . . . . . . . 55 Hydrolysis Rates of Acetals . . . . . . . . . . 60-75 DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . 76 Lysozyme-substrate Complex. . . . . . . . . . . 76 Trapping Experiments. . . . . . . . . . . . . . 78 iv TABLE OF CONTENTS--continued Page Hydrolysis of Acetals and Tetrahydropyran Derivatives. . . . . . . . . . . . . . . . 80 Hydrolysis of the Acetal Derivative . . . . . . 81 Rate Constants for Hydrolysis of the Tetra— hydrOpyran Derivatives . . . . . . . . . . 82 REFERENCES..................... 94 TABLE II. III. IV. VI. VII. VIII. IX. .XI. LIST OF TABLES Amino Acid Composition of the Enzyme-substrate Complex after Complete Enzymatic Hydrolysis. . Amino Acid and Carbohydrate Composition of Peak I of the Gel- filtered Peptic Hydrolyzate of Enzyme- -substrate Complex. . . . . . . . . Amino Acid and Carbohydrate Composition of Peak I of the Gel-filtered Hydrolyzate from the Complete Enzymatic Hydrolysis of the Enzyme-substrate Complex . . . . . . . . . . . Amino Acid and Carbohydrate Composition of Peak I of the Gel-filtered Tryptic Hydrolyzate of the Enzyme-substrate Complex. . . . . . . . Amino Acid Composition of the EnzymeeSubstrate Complex after Sodium Chloride-Guanidine Hydro- chloride Treatment . . . . . . . . . . . . . . Rate Constants for Hydrolysis of Tetrahydro- pyranyl Acetate in the Presence of and in the Absence of Hydroxylamine . . . . . . . . . . . Hydroxamic Acid Formation in the'Reaction of Tetrahydropyranyl ACetate with Hydroxylamine . Rate Constants for Hydrolysis of Acetamido- acetaldehyde Diethyl Acetal. . . . . . . . . . Rate Constants for Hydrolysis of 5-[(Tetra- hydropyran-Z-ylloxy]-pr0pionamide. . . . . . . Rate Constants for Hydrolysis of 5-[ITetra— hydropyran-2—ylloxy]-pr0pionic Acid. . . . . . Rate-Constants.for Hydrolysis of 5-[(Tetrar hydropyran-Z-yl)oxy]-propionic Acid Methyl Ester. . . . . . . . . . . . . . . . . . . . . vi Page 48 49 50 51 52 55 54 60 61 62 65 LIST OF TABLES-—continued TABLE XII. XIII. XIV. XV. XVI. XVII. Rate Constants for Hydrolysis of 2-(Tetra- hydr0pyran-2-yloxy)-acetamide. . . . . . . Rate Constants for Hydrolysis of 2-(Tetra- hydropyran-Z-yloxy)-acetic Acid. . . . . Rate Constants for Hydrolysis of 2-(Tetra— hydropyran-Z-yloxy)-acetic Acid Ethyl Ester. Rate Constants for Hydrolysis of 6-Ethoxy— tetrahydropyran-2-carboxamide. . . . . . . Rate Constants for Hydrolysis of 6-Ethoxy- tetrahydropyran-2-carboxylic Acid. . . . . Rate Constants for Hydrolysis of 6-Ethoxy- tetrahydropyran-2-carboxylic Acid Ethyl Ester. vii Page 64 65 66 67 68 69 LIST OF FIGURES Page Compounds cleaved by lysozyme. . . . . . . . . 5 Species isolated from M, luteus cell walls after controlled lysozyme hydrolysis (3.18). . 4 Lysozyme activity after reacting with M, luteus cell walls in the presence of hydroxyl- amine. . . . . . . . . . . . . . . . . . . . . 56 Lysozyme activity after reacting with M, luteus cell walls in the presence of and in the absence of sodium borohydride. . . . . . . 58 Plot ofwthe log of the second-order rate con- stant for hydrolysis of 2-substituted tetra- hydropyran derivatives against 6T (79) . . . . 7O Plot of the log of the second-order rate con- stant for hydrolysis of 2-substituted tetra- hydropyran derivatives against GI (79) . . . . 72 Plot of the log of the second-order rate con- stant for hydrolysis of 6-ethoxy-2-substituted tetrahydrOpyran derivatives against GI (79). . 74 viii INTRODUCTION Approach of the Present Investigation Lysozyme (5.2.1.17 N-acetylmuramide glycosylhydrolase) is the most thoroughly characterized glycosidase at the present time. Although its three dimensional molecular structure has been well defined by x-ray analysis, its cata- lytic mechanism, i.e., the means by which it catalyzes cleavage of the polysaccharide molecule, remains hypotheti- cal (1,2). The purpose of this study was to clarify some aspects of this mechanism. Two approaches were used in this investigation. The first one concerned the lysozyme-catalyzed reaction itself. It involved a) the determination of the nature of an enzyme- substrate complex formed between lysozyme and soluble M, luteus cell wall material discovered by Rynbrandt (5), and b) the search for chemical reactions that would trap a glycosyl-enzyme intermediate possibly formed during the lysozyme—catalyzed cell wall hydrolysis. In the second approach the rate constants for hydrolysis of acetal model compounds were studied. The acetal model I compounds contain neighboring groups which resemble either the amino acid side chains at the lysozyme active site or the acetamido group on the substrate molecule. Since all lysozyme substrates are cyclic acetals, mechanistic information may be obtained from these model compounds. Structure of the Lysozyme Substrates ,Lysozyme catalyzes the hydrolysis of the 6-(1-4) linkage between N-acetylmuramic acid and N-acetylglucosamine residues found in the polysaccharide material of most bacterial cell wall; it also catalyzes hydrolysis of N-acetylglucosamine oligomers derived from chitin (4-8), B-linked benzyl glycosides of di-N-acetylchitobiose (9), B-aryl glycosides of di-N- acetylchitotriose (10,11), and B—aryl glycosides of tri-N- acetylchitotriose (12). Recently(15-17,22), several compounds with a structure in which the 2-N-acetyl substituent on the sugar ring is absent (Figure 1), have been reported as undergoing lysozyme- catalyzed hydrolysis. It would seem therefore that the sub- strate acetamido group is not essential for lysozyme activity. Evidence will be reviewed later, however, that shows that the lysozyme-catalyzed hydrolysis seems to go faster when the acetamido grouP is present than when it is absent from the substrate (13,17). The substrate used in the preparation of the enzyme- substrate complexes studied in the present investigation was CH20H H20H o * ?R R = partially de-N-acetylated \\U chitin oligomers (Ref. 17) OH RO NHg NAC CHZOH H20H * ° y-Qm O HO on OH R = OH, NAc (Ref. 15) NAc HgoH O \\0 (Ref. 16) HO OH AC H20H H20H . H20H ¢H20H O \ * 0 H 0 (Ref. 16) OH _ OH I\ OH \ HO N A C C OH OH NA CH20H CHZOH CHZOH H20H O * O OH 7 OH R NAC H H H CH3CHCOOH R = N-acetylglucosamine (Ref. 16) * Bond cleaved in the lysozyme-catalyzed reaction Figure 1. Compounds cleaved by lysozyme. CH20H CHQOH CH20H HEOH O . O OH . OH O OH HO I . Ac 1 Ac NAc CH3CHCOOH CHaCHCO L-Ala I D—y-Glu-a-GlyCOOH L- ys-e-NHg GP-2 D-AlaCOOH HZOH CH20H O OH HO OH CH3CHCO fin NAc NAG L-Ala D—y-élu-a-GlyCOOH CH3CHCO L-Lys-e-NH . NH D-AlaCOOH L-Ala Day-Elu-arGlyCOOH L-Lys—e -NH2 D—Ala GP-l cnzoa CH20H HZOH H20. 0 OH 0 O . O OH 9 Hexasaccharide (R = CH3CHCOOH) Figure 2. Species isolated from M. luteus cell walls after controlled lysozyme hydrolysis (3,18). a glyc0peptide (GP-2) obtained from M, luteus cell walls by Rynbrandt (3) and Sharon and co—workers (18). Fraction I (5) was.also used. Fraction I is a mixture of oligosac- charides (mainly hexasaccharide) and glyc0peptides (mainly GP-1). The structure of these Species is shown in Figure 2. Lysozyme Active Site Structure The three-dimensional structures determined by x-ray studies of lysozyme and lysozyme-inhibitor complexes indicate that the active site of the enzyme is a cleft which is only half filled when tri-N-acetylglucosamine is an inhibitor. A total of six sugar residues can be fitted into this cleft in a satisfactory manner. Several specific interactions between the sugar residues and the protein molecule can be seen in such a structure. Each sugar residue occupies a "subsite“ in the cleft, and the six subsites have been desig- nated A, B, C, D, E and F reSpectively (19.20.21). By comparing the products formed from the hydrolysis of various chitin and cell wall oligomers, it has been concluded that the cleavage in the lysozyme reaction occurs between the D and E subsites (22,25,24). Model building indicates that sugar residue D makes reasonable contacts with the atoms of the protein molecule except that its CHZOH group makes too close a contact with the main chain C0 of residue 52, with Trp 108, and with the acetamido group of sugar residue C. This overcrowding can be relieved by distortion of the ring to a half-chair confor- mation. Inapection of the environment of subsite D shows that the side chains of Glu 35 and ASp 52 are disposed on each side of the 6-(1-4) linkage between the D and E residues, suggesting that these residues are involved in the cleavage mechanism. The carboxyl group of Glu 55 lies in a predomi- nately nonpolar region which makes it likely to be the carboxyl group postulated to have an abnormally high pKa (approximately 6.5) in the enzyme (25). The carboxyl group of ASp 52 lies in an essentially polar region and appears involved in a complex network by hydrogen bonds. This situ- ation may hold the residue in an ionized carboxylate state. It is interesting to note that the lysozyme-catalyzed hydrolysis of triéN-acetylglucosamine (24) and some aryl- glycosides (15) appears to have an absolute dependence of activity on a carboxyl group of normal pKa which probably is that of Asp 52. Postulated Reaction Mechanisms By putting to use the information obtained through this static picture of the lysozyme-substrate complex and the information about the reaction mechanism of the nonenzymatic hydrolysis of acetal and glycoside compounds, it is possible to prOpose probable mechanisms for the lysozyme-catalyzed reaction (28). All these mechanisms have in common the following steps: 1.-—Attachment of the substrate with simultaneous con- formational changes of the sugar residue D. This distortion brings the sugar ring part way to the transition state con- formation, thus decreasing the activation energy and conse- quently accelerating the reaction (29). 2.--Cleavage of the substrate linkage between residues bound at subsites D and E. This is accomplished by partici- pation of the Glu 55 carboxyl group. Since this carboxyl group probably is protOnated in the pH region of maximal lysozyme activity it could act as a general acid catalyst to protonate the glycosidic oxygen atom causing heterolysis of the carbon-oxygen bond. 5.-—The portion of the substrate lying in the E subsite and beyond is released from the cleft, leaving a glycosyl— enzyme intermediate. 4.-—The glycosyl-enzyme intermediate reacts either with water (hydrolysis reaction) or with an acceptor mole- cule (transfer reaction). It is necessary to postulate a glycosyl-enzyme inter- mediate because in all the lysozyme-catalyzed reactions studied so far, the stereochemical result is retention of the configuration at the C-1 atom of the sugar residue lo- cated in subsite D (15,16,26,50). For the cleavage step, which involves acid catalysis by Glu 55, the following mechanisms have been considered (15,28): 1.--The carboxylate anion of ASp 52 may act as a nucleo- phile, displacing the aglycone portion of the substrate and forming a mixed ester-acetal linkage in the glycosyl—enzyme intermediate. However, insPection of the model shows that the nucleophile and the leaving group cannot assume the cor- rect geometry for the transition state of a bimolecular sub- stitution. Furthermore, the carboxyl group of ASp 52 cannot, ’without considerable distortion, be located closer than 5 A. This distance would not allow the formation of a covalentv bond. 2.-~An alternative mechanism involves heterolysis of .the carbon-oxygen bond under the influence of the negative charge of the Asp 52 carboxylate anion. The reaction would proceed as it does in nonenzymic hydrolysis, by the formation of a carbonium ion. In solution the carbonium ion is stabilized by interaction with the dipolar molecules of the solvent, whereas in the lysozyme-catalyzed reaction the carbonium ion is stabilized by the negative charge held at a distance of aboutv5'A. 5.--The Asp 52 carboxylate anion may act as a general base by abstracting the proton of the substrate acetamido nitrogen with a simultaneous diSplacement of the aglycone. Examination of the lysozyme-substrate complex indicates that the Asp 52 participation in that fashion is not sterically feasible (2). 4.--It has also been prOposed that intramolecular nucleo- philic displacement of the aglycone portion of the substrate by the substrate acetamido group takes place forming a protonated oxazoline (28,40-42). However, it has been shown that the substrate acetamido group is not essential for enzymatic hydrolysis of the substrate. These possible reaction mechanisms are open to experi- mental test. There is the suggestion that the glycosyl-enzyme inter- mediate may resemble a carbonium ion more than a covalent intermediate (51,87): however, it would be premature to rule out of consideration a covalent mechanism in which Asp 52 may be involved (2). That is, conformational changes in the enzyme may take place in solution which can make sterical- ly possible the Asp 52 carboxylate nucleophilic participa- tion. In fact, the following observations may indicate that the lysozyme molecule under the influence of large sub- strates can undergo conformational changes: a) The chemical modification of ASp 87, Asp 66 and Glu 7 is considerably affected by the presence of substrate. These amino acid residues are not, according to the lysozyme-substrate complex model, in the cleft or in direct contact with the substrate (55). b) The carboxyl grouP of AsP 101 has been postulated to be bound to the acetamido nitrogen of sugar residue A and the O of C-6 of sugar residue B. In solution, however, the substrate apparently provides little protection for chemical modification of this asPartic acid chain and 10 moreover its chemical modification does not eliminate enzymatic activity against cell walls (55). c) The Trp 108 has been converted to a N'-formyl-kinurenine residue without any loss of either the catalytic activity or the binding capacity of lysozyme. .Since Trp 108 seems to be involved in the binding properties of the enzyme this finding indi— cates that the N'-formyl-kinurenine residue may act as well as the indole ring in the 108 position (54). Alternatively this result may indicate that Trp 108 is unimportant for binding the substrate in solution. Chemical Studies with Glycosidase Model Compounds Although no enzymatic reaction mechanism has been eluci- dated, it has usually been assumed that enzymes follow the same chemical principles that Operate in organic nonenzymatic reactions. In this sense the knowledge of the substrate cleavage position and the knowledge of the amino acid side chain composition in the lysozyme catalytic site, has stimulated basic research on the hydrolytic reaction mechanisms of ketal, acetal and glycoside compounds. Several independent lines of evidence (55) suggested that the acid-catalyzed hydrolysis of acetals and ketals, in dilute acid solutions, proceeds by a pathway (A-1) involv- ing a protonation step followed by a unimolecular rate deter- mining decomposition to an alcohol and a carbonium ion intermediate. 11 ng\_ ,IOR H0 R1‘\‘ ‘/,OR R1\\‘ ’/,OR /C\ ‘ /C\9 I /Ce +ROH R2 OR R2 OR R2 H according to the rate law k =k (H+)+ 2k (HA) obs H+ HAi i in which the terms in the summation on the right hand side of the equation are neglected. That is to say, general acid catalysis is insignificant compared to the specific acid catalysis. Recent findings, however, suggested that in acetal com- pounds in which the protonation step is more difficult or in which the A-1 transition state is sterically unfavorable, solvent participation and general acid catalysis can occur in the C-0 bond breaking step. These studies involved a series of 2-aryloxytetrahydr0pyrans (56), and 2-(pararsub- stituted phenyl)-4,4,5,5-tetramethyl-1,5-dioxolanes (57). The acid-catalyzed hydrolysis of glycopyranosides has been found to proceed by two reaction mechanisms (27,28). 1.—-Nuc1eophilic unimolecular substitution (A-1) in which, as in simple acetals, a carbonium ion is formed. 12 HQOH A H won HO (HOR) 5 HO -—~o CH20H / 9 H e CH20H / OH (HOR) Product HO OH H CH OH CH OH / 2 2 Q H OH B ( ) ——->- e (HOR) H H HOR H H OH ’ H The acid-catalyzed hydrolysis of glycosides usually involVes the formation of a cyclic carbonium ion (pathway A). This process requires a conformational change towards the half chain conformation (52) and hydrolysis is, character- istically, slower than in the case of simple acetals and ketals (28). 2.—-Nucle0philic bimolecular substitution (A-2) in which the bond making and bond breaking processes occur simultane- ously and the net stereochemical result is inversion of the configuration at the reaction center. The nucleophile (N) may be a solvent molecule or an intramolecular neighboring group CHgoH HO . H0 A m ’ HW“ —>' M3?! -+ “OH OH 15 It seems probable that lysozyme and other glycolytic enzymes function by extremely facile general acid-general base catalysis and, perhaps, molecular distortion mechanisms in which the participant groups catalyze the reactions near neutrality at rates much greater than can be observed in simple acid- catalyzed reactions at the same pH value. The observation of solvent participation and general acid cataly- sis, already mentioned, plus the neighboring group participa- tion, summarized in the following paragraphs, seems to be important for understanding the lysozyme-catalyzed reaction. Anchimeric assistance by the acetamido group in the Specific acid catalyzed hydrolysis of B—N-acetylglucosaminyl bonds has been reported by Bruice et al. (40-42). This assistance showed a remarkable dependence on the nature of the aglycone. .The most probable mechanism was thought to be a nucle0philic diSplacement by the 2—acetamido oxygen of the protonated aglycone. (A similar mechanism had been previously proposed for the hydrolysis of acetylated glycoside halides (28) in which an oxazoline intermediate is formed. CHgOH H CH20H [ion 8 "ROH ' 9 HO OH \ 9 HO OH \Z —+ roduct HN‘W //0 ’/ NCHa “ 6 H3 Isolated cases of intramolecular carboxyl group participa- tion has been observed in glycosides. Karrer (44), Helferich 14 (45), Capon (46). and Bruice (41,47) observed that o-carboxy- phenyl-B-D-glucoside is spontaneously hydrolyzed in aqueous solution. Three possible mechanisms have been hypothesized (intramolecular general acid catalysis, intramolecular dis- placement of the protonated aglycone by carboxylate ion, and intramolecular nucleophilie-electrophilic catalysis) for this process (46,47). The carboxyl group participation in the hydrolysis of poly- and oligouronides has been proposed to involve general acid catalysis (48,49). Evidence for car- boxyl group participation in the hydrolysis of methyl-d-D- gluc0pyranosiduronic acid has also been presented (50). .There is less information for carboxyl group participa- tion in acetal and ketal hydrolysis than that for glycosides. Actually only one case has been reported, that by Capon (51). This investigator interpreted the enhanced hydrolysis rate of o-methoxymethoxybenzoic acid as an intramolecular acid catalysis CHg-O-CHa CH2 I 2Z3 __.). +0 |o —>- Products . 2 l \\TI//(S CH3 Bruice and Piszkiewicz (47) in an attempt to study the carboxyl group participation, found that there is no carboxyl group participation in the hydrolysis of seven 1,5-dioxanes and 1,5-dioxa1anes. 15 Chemical_Studies on thegLysozyme—Catalyzed Reaction Mechanism Several different approaches have been used in the past to determine the participation of individual amino acid side chains in the catalytic activity of lysozyme. Most of these approaches involved chemical modification of the amino acid side chains. Thus, esterification with methanol-hydrochloric acid of carboxyl groups (55), acetylation of amino groups (54), iodination of tryptophan (55), oxidation (55) or reduc- tion (57) of disulfide bonds, photooxidation of histidine and aromatic amino-acids (58) and oxidation of tryptOphan (56) cause partial or complete inactivation of lysozyme. The significance of these investigations with reSpect to the catalytic role of the modified amino acid is difficult to assess since the chemical modifications were not associ- ated with either the binding properties or the catalytic properties of the enzyme. Most recently, however, many of these limitations have been overcome. The availability of soluble, low molecular weight substrates of known mole— cular structure have permitted the use of techniques that can differentiate between binding properties and catalytic properties. Furthermore, the information about the three dimensional structure of the lysozyme-inhibitor complexes_ and the conclusions inferred from model building, have directed most of the chemical modification studies to a limited number of amino acid residues. 16 Carboxylic acid side chains have been the most thorough- ly studied, not only because of the postulated role in lysozyme, but also because of the earlier postulation of their catalytic participation in other glycosidases, d-amylase (59) and B-glucosidase (60). In only one instance, however, has the direct participa- tion of a carboxyl grOUp in lysozyme catalysis been reported (61). »A single carboxyl group was esterified with triethyl— oxonium fluoroborate. The lysozyme derivative was isolated from other esterified lysozyme Species and showed to have essentially no enzymatic activity against E, luteus cell walls, whereas it retained good substrate binding prOperties. The carboxyl group was identified as Asp 52. The results from other recent modifications of the car- boxyl side chains in lysozyme confirm their necessity for activity but do not prove or diSprove their catalytic role. Thus, Kravchenko (62) and Raftery (65) have carried out esterification using methanol-hydrochloric acid. Binding studies (65) indicated that the tertiary structure in the region of the binding site was not seriously disrupted by the esterification process. The activity of the esterified enzyme was, however, 5 to 5%iof that of native lysozyme. Koshland et al. (55), using a newly developed technique have modified in the presence of substrate all the carboxyl groups except those of Glu 55 and Asp 52. The modified lysozyme retained 60%Lof the native enzymatic activity. 17 Removal of the substrate led to modification of all the car- boxyl groups except that of Glu 55; this resulted in loss of activity. The participation of Asp 52 in binding prOper- ties or catalytic prOpertieS was not investigated. Chemical modification of lysine (65), histidine (65), and tyrosine (65) side chains have shown that they are not implicated either in binding prOpertieS or catalytic prOper- ties in the lysozyme catalyzed reaction. These results agree with the conclusions obtained from x-ray studies. A different approach for studying the implication of amino acid side chains in the catalytic activity of lysozyme has been undertaken by Sharon and co-workers (64). These investigators designed a Specific inhibitor of lysozyme which can react with carboxyl groups. CHZOH 0—CH2 "CH—CHg / 0 HO OH \ Ac Incubation of this compound with lysozyme produced a cos valently bound enzyme-inhibitor complex. The alkylated amino acid residue was not identified. Another approach was originated in this Laboratory by Rynbrandt (5). The enzyme was incubated with low molecular weight substrates (Figure 2) obtained from g, luteus cell- walls and the reaction was quenched with guanidine hydro- chloride. After reduction of the disulfide bonds and alkyla- tion with N-ethylmaleimide a stable lysozyme substrate complex 18 was obtained. The author suggested that these may be covalently bound complexes formed as intermediates in the lysozyme-substrate reaction. There is also the possibility, however, that these com- plexes are the result of electrostatic binding of the sub- strate to the enzyme, Since the lysozyme molecule is posi- tively charged and the substrate negatively charged under the conditions used for preparing the complex. In fact, Rynbrandt (5) observed that the complex does not form when the substrate is freed of pentapeptide, i.e., when the nega- tive charge is decreased. Also the complex does not form when the sulfhydryl groups of the reduced enzyme are alkyl- ated with iodoacetate, which increases the negative charge on the protein. .Finally, it was found that previously in- activated lysozyme also combines with substrate, though to a less extent than it does when the lysozyme is denatured and chemically modified in the presence of substrate. In spite of these observations, the possibility of a covalent complex still exists. Thus, part of the present work was to investigate the nature of this complex. Substrate Acetamido Group Participation It is evident from the nature of different substrates_ lysozyme can hydrolyze that the acetamido group at C92 of the sugar residue D is not essential for lysozyme activity (Figure 1). H0 19 There are, however, two instances in which an enhance- ment effect of the substrate acetamido group in the rela— tive rate of the lysozyme-catalyzed reaction has been ob- served. One case involved the comparison of the hydrolysis rate of the indicated bonds of compounds I and II (15). Compound I hydrolyzed 102 times faster than compound II. These results however, Should be considered tentative since obtaining the relative values involved several assumptions (2). H20H CH20H @Qfiow has in. Lowe and Sheppard (15) interpreted these results as evidence for a lysozyme mechanism in which general acid catalysis by Glu 55 is assisted by the acetamido group. Another study was carried out with 50% de-N-acetylated chitin oligomers (17). Although these substrates bind to the enzyme as well as the unmodified chitin oligomers, the hydrolysis rate of the de-N-acetylated ones was only 20% . of that of the normal chitin oligomers. Hayashi and co- workers (17) postulated that the positive charge of the amino group left instead of the acetamido group is responsible for the decrease in enzymatic activity. EXPERIMENTAL Reagents General All concentrated acids, common inorganic salts and organic reagents were reagent grade. Lygggyme_Reagents 1. Amberlite MB-5 mixed bed resin, 20-50 mesh Rohm and Haas 2. Amberlite CG-120 resin Amberlite CG—120 type II (200 mesh and finer) purchased from Rohm and Haas had previously been classified (5) for quantitative amino acid chromatography by the method of Hamilton (89); the fraction used in this work was the coarse residue which did not flow at a wash velocity of 590 ml per min. 5. Aminex AG 50W-X2, 200—500 mesh, hydrogen form .California Corporation for Biochemical Research and BioRad Laboratories. 4. Biogel P-4, 50-100 mesh BioRad Laboratories 5. Carboxymethyl Cellulose California Corporation for Biochemical Research 20 6. Dialysis tubing "Visking" sausage casing (18/52 in), purchased from Union Carbide Corporation. 7. Ethanol assay system Determatube C-ALK ethanol assay system purchased from WOrthington Biochemical Company. 8. Glass beads Minnesota Mining and Manufacturing Company “superbrite" type 150-5005 0.1 mm diameter glass beads were stirred in hot chromic acid cleaning solution for Six hours, then ex- haustively washed with distilled water and dried at 105°. 9. Guanidine hydrochloride Reagent grade guanidine hydrochloride (Mallinckrodt) 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 white solid was recrystallized from 95% ethanol. 10. Hydroxylamine hydrochloride Matheson, Coleman and Bell (practical grade) or Mallinckrodt (reagent grade) recrystallized from half of its weight in water-95% ethanol (4:1, v/v). 11. Leucine aminopeptidase Leucine aminopeptidase was prepared by Dr. J. C. SpeCk according to the method of Hill 25 al. (90). The enzymatic activity (C1 = 5) of the preparation used in the present 22 work was determined by the method of Bryce and Rabin (91). 11. Lysozyme Worthington Biochemical Corporation twice recrystal- lized salt-free lysozyme and Sigma Chemical Company three times crystallized salt-free lysozyme. 12. Micrococcus luteus cells, dried Sigma Chemical Company pfs grade, and Miles Laboratories, Inc. 15. PepSin Pentex Incorporated three times crystallized pepsin. 14. Prolidase Prolidase was purified from swine kidney by the method of Davis and Smith (92). The preparation which is obtained at Step 2 (C1 = 10 to 20) was used. 15. Papain Papain, twice crystallized suspension (55 mg/ml, 15 u/mg). Worthington Biochemical Corporation. 16. Sephadex G-15 Pharmacia Fine Chemicals, Incorporated. 17. Trypsin Wbrthington Biochemical Corporation twice recrystal— lized salt-free trypsin. 18. Urea Mallinckrodt reagent grade. Urea solutions were de-‘ ionized with Amberlite MB-5 mixed bed resin, filtered and recrystallized from the concentrated water solutions. 25 Acetal!ReagentS 1.OQm .< ca omumoflpcfl mm wmmmm >ua>auom ms» How cmxmu muw3 muosvflam pom coflusHom mcHEmawxoupmc SH Uw>aommH© mm3 mE>N0m>H .Houucoo m m¢II.m 0mm owa .¢IIIIIIIV >DH>Huum wfimuomma How omwmmmm pom SHE .owa pom Odd .0m .0 um cmxmm Muwz muosqfiafi .mmuocHE oma How Ummooum ou pw3oHHm cofiuommu map can mafia? Hamo.mmwmmfl rm nufl3 Umoom mm3 SOHDSHOm mcflemexouomz SH Um>aommflp mE>Nom>qll.¢ "30Hwfl .m Ucm d CH Umumoflocfl mm Uwumwuu wemuomwa m0 m1 m pom Acoflusaom mpflHoHno Esflpom ucmonmm a mo HE OH pom .Hmmmsn mwmnmmosm EDHUOm m.m mm .2 emo.o mo HE om SH Uwpcmmmsm mHHmU pmfiup m0 m8 mav coflmcmmmom Hawo mmwmmfl.rm mo HE o.m omcflmucoo mmmmm 30mm .wcflEmmeouoms mo wocmmmum may cfl mHHm3 Hawo msmuoa .m.£ua3 mcfluommu Hmumm >ua>auum wEmuoqu .m musmflm 57 m musmflm ww...:z:z on. 0 0_ On — W ‘ Amy Jomkzoo I 3: 20_h0ua>fluom memuomwa How omwmmmm 0cm cmxmu meB muooqflam .mEHu cofluomMHaommwo mfiwuowwal.¢ "BonQ .m 0cm m cm Umumoflocfl mm Umummuy mewuomwa mo 01 m 0cm ACOHDSHOm mUHHoHLU ESHUOm ucwoumm H mo HE 0a 0cm .ummmsn mumnmmonm Esapom m.m mm .2 >00.o no He 0m SH poocmmmom maamo Umfluo mo 08 mdv coflmcmmmsm Hamo mmmmmfl.flfl mo HE 0.m Umcflmucoo mnouxHE hmmmm 30mm .mpflnpwnouon EDHUOm wo mocmmflm may SH 0cm mo wocmmwum may CH mHHMB Hamo msmuoa 2% nuHB mcfluomwu Hmumm >Dfl>fluom mE>NOm>A .d whomflm 59 00. e musmflm $52.: on 8 100.0 9 .\ .\¢ m v 0 V S. E 100.0 .8 .5520... .. :3 2253.... . oo._ Hydrolysiszates of Acetals 60 Table VIII. Rate Constants for Hydrolysis of Acetamido- acetaldehyde Diethyl Acetal» The reaction mixture contained 2 mmoles<fifacetamidoacetalde- hyde diethyl acetal, 10.0 ml of dioxane and 10.0 ml of perchloric acid or buffer solution. .The change in absorbance was measured at 280 mu. Perchloric Acid, M 104k; 104k; 0.082 0.164 0.78 9.4 0.082 0.528* 0.78 9.4 0.10 0.20 0.77 7.7 0.10 0.20 0.77 7.7 0.165 0.550 1.7 10.0 0.165 0.550 1.7 10.0 0.1 M, pH 4.0 acetate buffer 0.00 0.2 M, pH 8.9 Tris buffer 0.00 * Ionic strength adjusted with potassium bromide. 61 Table IX. .Rate Constants for Hydrolysis of 5-[(Tetrahydro- pyran-2-yl)oxy]—pr0pionamide The reaction mixture contained 4 mmoles of the amide, 10 ml of dioxane, and 10 ml of perchloric aci absorbance was measured at 280 mu. d. The change in Perchloric Acid, M 103k; 102k2 0.025 0.44 1.75 0.012 0.22 1.75 0.098 2.2 2.2 0.10 1.95 1.95 0.10 2.05 2.05 Average 1.94 62 Table X. Rate Constants for Hydrolysis of 5-[(Tetrahydro- pyran-2-yl)oxy]-propionic Acid To prepare the free acid form of this compound, 0.01 mole of- the correSponding methyl ester was weighed in a 25 ml volu- metric flask. Then, 0.01 mole of sodium hydroxide in s01ution and an equal volume of dioxane were added. After the Saponi- fication reaction had been completed it was diluted to 25 ml with 50% dioxane-water (v/v). For the kinetic runs 10.0 ml of this solution and 10.0 ml of perchloric acid i8 50% dioxane-water, previously equilibrated at 25.0.: 0.1 , were mixed and transferred to the Spectrophotometric cells. Perchloric Acid, M 103k; 102k2 0.10 1.95 1.95 0.10 1.95 1.95 0.10 2.05 2.05 0.10 2.05 2.05 Average 2.0 pH 4.8* 0.00 0.00 * This pH value was obtained by half neutralization of the salt formed in the saponification reaction. 65 Table XI. .Rate Constants for Hydrolysis of 5-[(Tetrahydro- pyran-2-yl)oxy]-prOpionic Acid Methyl Ester The reaction mixture contained 4 mmoles of the ester, 10.0 ml of dioxane, and 10.0 ml of perchloric acid. The change in absorbance was measured at 280 mu. Perchloric Acid, M 104k; 102k2 0.05 4.95 0.99 0.05 4.95 0.99 0.05 4.90 0.99 0.05 4.90 0.99 Average 0.99 64 Table XII. Rate Constants for Hydrolysis of 2-(Tetrahydro- pyran-2-yloxy)—acetamide The reaction mixture contained 4 mmoles of the amide, 10.0 ml of dioxane, and 10.0 ml of perchloric acid. .The change in absorbance was measured at 280 mu. Perchloric Acid, M 103k; 102kg 0.10 5.0 5.0 0.10 2.9 2.9 0.10 5.0 5.0 65 Table XIII. -Rate Constants for Hydrolysis of 2-(Tetrahydro- pyran-2-yloxy)-acetic Acid To prepare the free acid form of this compound, 0.01 mole of the corresponding ethyl ester was weighed in a 25 ml volu- metric flask. Then, 0.01 mole of sodium hydroxide in solution and an equal volume of dioxane were added. “After‘the'saponi~ fication reaction had been completed it was diluted to 25 ml with 50% dioxane-water (v/v). For the kinetic run 10.0 ml of this solution and 10.0 ml of perchloric acid i 50% dioxane—water, previously equilibrated at 25.0.: 0.1 , were mixed and transferred to the SpectrOphotometric cell. Perchloric Acid, M 103k; 102k2 0.10 6.1 6.1 0.11 6.5 5.9 0.11 7.0 6.4 0.11 7.6 6.9 0.11 7.7 7.0 0.10 5.9 5.9 Average 6.4 pH 4.8* 0.00 0.0 * This pH value was obtained by half neutralization of the salt formed in the saponification reaction. 66 Table XIV. Rate Constants for Hydrolysis of 2-(Tetrahydro- pyran-2-yloxy)-acetic Acid Ethyl Ester The reaction mixture contained 4 mmoles of the ester, 10.0 ml of dioxane, and 10.0 ml of perchloric acid. The change in absorbance was measured at 280 mu. Perchloric Acid, M 103k; 102k2 0.097 5.5 5.4 0.097 5.2 5.5 0.097 5.4 5.5 0.10 5.8 5.8 67 Table XV. Rate Constants for Hydrolysis of 6-Ethoxytetra- hydropyran-Z-carboxamide The reaction mixture contained 4 mmoles of the amide, 10.0 ml of dioxane and 10.0 ml of perchloric acid. .The change in absorbance was measured at 280 mu. Perchloric Acid, M 105k; 104k2 0.05 2.4 4.8 0.10 5.0 5.0 0.10 6.0 6.0 Average 5.5 68 Table XVI. .Rate Constants for Hydrolysis of 6-Ethoxytetra- hydropyran-Z-carboxylic Acid To prepare the free acid form of this compound, 0.01 mole of the corresponding ethyl ester was weighed in a 25 ml volumetric flask. Then, 0.01 mole of sodium hydroxide in solution and an equal volume of dioxane were added. 'After the saponification reaction had been completed it was di- luted to 25 ml with 50% dioxane-water (v/v). For the kinetic run 10.0 ml of this solution and 10.0 ml of perchoric acid in 50% dioxane-water, previously equilibrated at 25.0.: 0.10, were mixed and transferred to the SpectrOphotometric cell. ,Perchloric Acid, M 105k; 104k2 0.10 5.6 5.6 0.10 5.2 5.2 0.10 5.5 5.5 Average 5.4 pH 4.8* 0.00 0.00 *- ThiS pH value was obtained by half neutralization of the salt formed in the saponification reaction. 69 Table XVII. Rate Constants for Hydrolysis of 6-Ethoxy- tetrahydropyran-Z-carboxylic Acid Ethyl Ester The reaction mixture contained 4 mmoles of the ester, 10.0 ml of dioxane, and 10.0 ml of perchloric acid. The change in absorbance was measured at 280 mu. Perchloric Acid, M 105k; 104k2 0.10 5.5 5.5 0.10 . 4.8 4.8 0.10 4.8 4.8 Average 5.0 70 .AmnyHo umcflmmm mm>Hum>HHm© cmuwmouv>£muumu Uwuouflumnsmlm mo mflmwaouvhn Mom ucmumcoo mums umUHOIpcoomw wnu mo 00H 0:» mo uon .m mnsmflm 71 000 m mnzmflm .0 0N0 0.0 00.0 0.00 .9..- .I m m a :o co 5.. :Noomzo .o.~...M O u" mzzoomzo 616 z 10...... 6.0 mmxulo o 72 H .Amcv c umcecmc mm>Hum>HHmU cmuwmouvwnmnumu Umusuflumnomlm mo mammaoupmn Mom ucmumcoo mums HmUHOIGSOUmm 0:» mo moH mzu mo poam .0 musmflm 75 000 0N0 m musmflm 020 000 .mmoo «:23 J 000! 40h! lwhwl mzo 0 :«oo care C) 0;! 00 3» 601 74 .Amwv Ho umcflmmm mm>flum>flumw cmuwmoupmcmuumu Umusuflumnsmiml%xonumum mo mflmwaoucwn How ucmumcoo mums umUHOIUcoomm 05D mo 00a mnu mo uon .w mnsmflm ‘ 75 0nd 0N0 q n musmflm 0:0 000 000- DISCUSSION AND CONCLUSIONS Lysozyme-substrate Complex The lysozyme-substrate complex discovered by Rynbrandt (5) was investigated to determine the nature of the binding between the enzyme and the substrate. The preparation of the enzyme-substrate complex in- volved: 1) incubation of the substrate with lysozyme for 15 minutes, 2) quenching the reaction by disrupting the three- dimensional structure of lysozyme with guanidine hydrochloride, 5) reduction of the disulfide bridges and 4) permanent lyso- zyme inactivation by alkylation of the liberated thiol groups with N-ethylmaleimide. The presence of substrate in the complex was verified by amino acid analysis. The dialyzed and washed complex was subjected to peptic hydrolysis, and the hydrolyzate then analyzed by gel filtra- tion. A peak which contained only substrate material was found (Table II). .The same result was obtained when the complete enzymatic hydrolysis procedure of Hill and Schmidt (75) was used (Table III). These observations were inter- preted as evidence that the Substrate was not covalently bound to the enzyme Since it becomes free after the protein 76 77 is destroyed. An alternative eXplanation, however, is that the enzymatic treatment may have cleaved the covalently bound substrate. This alternative explanation can be valid for both the peptic and the complete enzymatic hydrolysis, since the Specificity of these enzymes iS broad. AS a milder procedure for isolating a peptide-substrate complex, tryptic digestion of the enzyme-substrate complex was carried out. Gel filtration of this enzymatic hydroly- zate gave a peak which contained both substrate and a mix- ture of peptides. However, a control consisting of a mix- ture of substrate and tryptic peptides from reduced and N—ethylmaléimide—-treated lysozyme yielded a similar peak upon gel filtration (Table IV). Therefore, it is evident that the peptide-substrate complex, if any, was mixed with other peptides and therefore required further separation studies. A different approach, however, confirmed the supposi- tion that the complex was formed through electrostatic interactions and not through covalent binding. It was dis- covered that washing the enzyme-substrate complex (dissolved in 6 M guanidine hydrochloride) with 1 M sodium chloride completely eliminated the substrate from the protein. This was accomplished at pH 4, 7 and 9 (Table V). These results indicate that the substrate is not covalently bound to the enzyme. 78 Trapping EXperiments According to postulated mechanisms, Asp 52 may be in- volved in the glycosyl-enzyme intermediate (a) in the lysozyme-catalyzed reaction. Another (77), but so far un- eXplored possibility is the participation of the Gln 57 amido group (b). 5.0%: . H ' O C=O 0 E =NH2 nzyme | Enzyme (a) (b) -Since hydroxylamine is a good trapping reagent for esters an attack on the acylal intermediate at the carboxyl carbon by hydroxylamine iS reasonable. During the course of this investigation, however, it was observed that lysozyme is not inactivated during the reaction with M, luteus cell walls in the presence of 1 M hydroxylamine (Figure 1). This lack of inactivation was nevertheless inconclusive, since the reaction of hydroxylamine with acylals had not been in- vestigated. Tetrahydropyranyl acetate was used as a model compound for studying the hydroxylamine reaction with acylals. .The results of these eXperiments (Table VII) Show that the 79 reaction with hydroxylamine forms only about 5 percent of hydroxamic acid during the time required for tetrahydro- pyranyl acetate to undergo complete hydrolysis. On the other hand, the kinetic eXperiments designed to determine the reactivity of hydroxylamine towards the acetal center of this model compound (Table VI) Show that the rate con- stant for hydrolysis has the same value with or without hydroxylamine in the reaction mixture. Thus, it can be con- cluded that the low reactivity of hydroxylamine towards the ester center is not due to a preferential reactivity towards the acetal center but rather to a competition between hydrolysis of the acetal bond and nucleophilic attack at the ester carbonyl carbon by hydroxylamine. Since the glycosyl-enzyme intermediate (a) is eXpected to be rapidly converted into the carboxyl form, hydroxyl- amine attack at the ester center would not presumably be favored during the lysozyme-catalyzed reaction. It was also of interest to study the effect of sodium borohydride on the catalytic activity of lysozyme. -Recent studies have shown that cyclic imidates can be reduced to aldehydes with sodium borohydride (76). If an imidate glycosyl-enzyme intermediate (b) were present in the lysozyme- catalyzed reaction and if reduction of this intermediate could be accomplished, then inactivation of the enzyme would occur. Inactivation, however did not happen when lysozyme was reacted with M, luteus cell walls in the presence of sodium borohydride (Figure 4). 80 To see if an acyclic imidate can actually be reduced by sodium borohydride, two model compounds were studied, methyl acetimidate and ethyl acetimidate. The results of these studies Show that neither one is reduced under the condi- tions employed (page 55). In summary, then, the eXperiments with the lysozyme itself indicate that the investigated enzyme-substrate com- plex is not a covalently bound complex. Electrostatic forces seem to be reSponsible for the strong association phenomenon. However, if a covalently bound glycosyl—enzyme intermediate is formed during the lysozyme-catalyzed reaction, an acylal or an imidate intermediate are not excluded by the results obtained with model systems. Hydrolysis of Acetals and TetrahydrOpyran Derivatives In the design of both the acetals and the tetrahydro- pyran derivatives studied here, the substituents were chosen such that each series of model compounds would allow a differ- entiation between intramolecular catalysis and inductive effects on the rate constant of the acid-catalyzed hydrolysis, if intramolecular catalysis was indeed present. The tetrahydrOpyran derivatives bear a marked resemblance to structures commonly associated with glycosides. It is known that substitution in both the aglycone and the sugar ring usually influences the hydrolytic rate constant of 81 glycosides in a manner dependent on the inductive effect of the substituent (78). In this reSpect, the tetrahydrOpyran derivatives would appear to be ideal model compounds with which to study the effect of the substituents in the glyco- side hydrolysis, free from complications created by adjacent hydroxyl groups. Hydrolysis of the Acetal Derivative The inductive constant, 0;, is similar for both the ethoxy and the acetamido group (79). Therefore the second- order hydrolysis rate constant for 2-ethoxyacetaldehyde diethyl acetal and acetamidoacetaldehyde diethyl acetal should be similar if other polar effects, resonance effects, and steric effects are Similar, as seems to be the case. Comparison of the second-order hydrolysis rate constants of the acetamidoacetaldehyde diethyl acetal (Table VIII) with that of the 2-ethoxyacetaldehyde diethyl acetal, Shows that, at the most, acetamidoacetaldehyde diethyl acetal hydrolyzes five times as fast if 2.0 x 10‘4 is taken as the unit value or at the same rate if 8.6 x 10‘4 is taken as the unit value. 1 CH3CHg—O-CH2CH(OCH2CH3)2 2-ethoxyacetaldehyde diethyl acetal k2 = 2.0 x 10’4 1. mole‘1 sec-1 (5) kg 8.6 x 10"4 1. mole‘1 sec’1 (80) 82 2 CH3CO-NH-CH2CH(OCH2CH3)2 2—acetamidoacetaldehyde diethyl acetal kg 9.0 x 10‘4 1. mole‘1 sec‘1 The structural position of the acetamido group in the acetamidoacetaldehyde diethyl acetal is Similar to that of the acetamido group in the B-N-acetylglucosaminyl deriva- tives studied by Bruice. Since the rate constants for the hydrolysis of these glycosides are higher than eXpected, Piszkiewicz and Bruice (40-42) have proposed that acetamido group participation can account for the rate-augmenting effect observed. The results obtained with acetamidoacetaldehyde diethyl acetal, however, indicate that there is little or no partici- pation by the acetamido group in the hydrolysis of this compound. This lack of participation may be due to entrOpy factors and is discussed later in connection with the tetra- hydropyran derivatives. Rate Constants for Hydrolysis of the TetrahydrOpyran Derivatives All of the prOpyloxytetrahydropyran-related deriva- tives hydrolyze at about the same rate: 5 ( >-O-CH3CH3 2—ethoxytetrahydr0pyran k2 = 1.6 x 10’2 1. mole‘1 sec"l 85 0 4 . o -CH2CH2CONH2 5—[(tetrahydrOpyran-Z-yl)oxy]-propionamide k2 = 1.9 x 10'2 1, mole‘1 sec-1 5 . . “CHQCH2COOH 5-[(tetrahydrOpyran-Z-yl)ony-propionic acid k2 = 2.0 x 10‘2 1. mole'1 sec‘1 6 -CH2CH2COOCH3 5-[(tetrahydropyran-Z-yl)oxy]-propionic acid methyl ester k2 = 1.0 x 10'2 1. mole”; sec-l Since the amide, carboxyl and ester substituents are three atoms away from the reactive center their inductive effects have only a small influence in the hydrolysis process. Furthermore, there appears to be no intramolecular catalysis from any of the substituents Since all their inductive con- Stants (79) agree very closely with the log of the reSpective rate constant value when a linear free-energy relationship of the type described by the Hammett equation log k = p6 84 is applied to the hydrolysis rate constants of this series of compounds (Figure 5). When the amide, carboxyl and ester substituents are positioned two atoms away from the center of hydrolysis, as in the case of the ethoxytetrahydropyran-related derivatives, the rate constants are Slightly affected: 5 *CHZCHg 2-ethoxytetrahydr0pyran k2 = 1.6 x 10---2 1. mole'1 sec--l . 7 .‘0 ‘CHgCONHg 2—(tetrahydrOpyran-Z-yloxy)~acetamide k2 = 5.0 x 10"2 l. mole‘l sec‘1 8 -CH2COOH 2-(tetrahydrOpyran-Z-yloxy)-acetic acid k2 = 6.4 x 10-2 1. mole'l sec‘1 9 -CH2COOCH2CH3 2—(tetrahydrOpyran~2-yloxy)~acetic acid ethyl ester k2 = 5.5 x 10'2 1. mole‘1 sec‘l 85 All of the rate constants are higher than that of the parent compound, 2-ethoxytetrahydr0pyran. These rate con- stants could not be eXplained on the basis of an intra- molecular participation of the amide and carboxyl groups, Since the ester substituted compound, which apparently can- not assist in the hydrolysis process, undergoes hydrolysis at about the same rate. .Moreover, the rate constants for hydrolysis of all the compounds in the series appropriately fit on a straight line after a Hammett type free-energy relationship is applied (Figure 6). The results with compounds 4 to 9 can best be explained by assuming that the hydrolysis proceeds via a cyclic car- bonium ion (pathway A), rather than an acyclic carbonium ion (pathway B) as follows: ‘99 __;> \ A v— \ + ROH rOdCS. H/ [{Iast 0R Product O H fagg§¢ B 0 —-‘ R <7.- r.d.S. The inductive effect of the amide, carboxyl, and ester sub- stituents would decrease the basicity of the exocyclic 86 oxygen and consequently decrease the equilibrium concen- tration of the protonated intermediate. But at the same time they increase the ease of carbon-oxygen cleavage. Partial cancellation of the Opposing effects Should have small influence on the hydrolytic rate constants. For the same reasons a p of small magnitude Should be observed in a Hammett type free-energy relationship plot. This actually was found (Figure 5 and 6). This would not be expected if an acyclic carbonium ion (pathway B) were involved, Since it would be destabilized by an aglycone having the inductive characteristics of the amide, carboxyl, or ester substit- uents. This destabilization would produce a decrease in the rate constants relative to the parent compound 2-ethoxy- tetrahydropyrah. A similar Situation is well documented for acyclic acetals (84) and for 2-alkoxytetrahydrOpyrans (85). Intramolecular catalysis in the hydrolysis of compounds 2, 4, 5, 7, and 8 was eXpected since the structural position of their acetamido, amide, and carboxylate groups are Similar to the corresponding acetamido and carboxyl groups in B—N- acetyl-glucosaminyl derivatives (40-42) and o-carboxyphenyl- B—D—glucoside (46). The rate constants for the hydrolysis of these glycosides are higher than eXpected. Piszkiewicz and Bruice (40-42) have prOposed acetamido group participation (a), and Piszkiewicz and Bruice (41) and Capon (46) have prOposed general acid catalysis (b) to account for the dif- ference observed. AS alternative mechanisms Capon (46) has 87 proposed nucleophilic diSplacement by the carboxylate group (c) or a combination of nucleophilic and electrophilic mechanisms (d): CH2 OH 32 H H H6). '9 (a) CH3 CHZOH He J H OH ( H0e (c) (d) However, because of steric effects, a nucleophilic dis- placement by the amide or the carboxylate groups in compounds 4, 5, 7, and 8 appears to be unlikely in the hydrolysis via a cyclic carbonium ion, as postulated here. In the orcarboxyphenyl-B-D-glucoside (41,46), as well as in all the compounds in which intramolecular catalysis by the carboxyl group has been reported, i.e., methyl-a-D- glucopyranosiduronic acid (86) and polysaccharides containing 1,4-linked hexuronic acids (48,49), the carboxyl group posi- tion is relatively well defined with respect to the center of hydrolysis. .The same can be said about the acetamido group in 8—N-acetylglucosaminyl derivatives (40-42) and in 88 the acetylated glycoside halides (28). It would seem there- fore that for the acetamido group of compound 2 to partici— pate in a nucleophilic displacement and for the carboxylic group of compounds 5 and 8 to participate as a general acid in the hydrolysis of these compounds, restrictions in the Spatial characteristics of the neighboring group and the reaction center are also required. In compound 2, 5 and 8, however, a restriction of this type does not occur since the aliphatic carbon chains allow a relatively large degree of freedom for the movements of the substituent groups. This conclusion appears to be supported by preliminary results obtained in this Laboratory with 2-(o-carboxyphenoxy)tetra- hydropyran, a compound in which the carboxyl group is in a restricted position Similar to that of the carboxyl group of o-carboxyphenyl-B-D-glucoside (46). 2-(o-Carboxyphenoxyl)- tetrahydropyran (I) has been found to hydrolyze at pH 7.4 with k0 = 1.9 x 10-3 sec-'1. bs 0 COOH (I) The results obtained with the 6-ethoxy-2-substituted tetrahydrOpyranS, compounds 10 to 12, indicate that the electron withdrawing character of the amide, carboxyl, and 89 ester groups decrease the rate constants of the acid- catalyzed hydrolysis relative to the parent acetal 2-ethoxy- tetrahydrOpyran, by two orders of magnitude: 10 11 12 0"CH2CH3 o 2-ethoxytetrahydropyran k2 = 1.6 x 10'2 1. mole‘1 sec‘l CONHg O-CH2CH3 Q 6-ethoxytetrahydropyran-2-carboxamide k2 = 5.5 x 10'4 1. mole-1 sec'l OOH O ‘CHgCHs 6-ethoxytetrahydropyran-2-carboxylic acid k2 = 5.4 x 10‘.”4 1. mole‘1 sec‘l COOCH2CH3 O‘CHgCHg CT 6-ethoxytetrahydropyran-2-carboxylic acid ethyl ester kg 5.0 x 10"4 1. mole-1 sec‘l These results are to be eXpected from previous studies on structurally similar compounds. For example, there is a 90 decrease, though to a smaller extent, in the acid-catalyzed hydrolysis rate constant of gluc0pyranosiduronides relative to the correSponding glucopyranosides (81). Also in some cyclic acetals [e.g., 1,5-dioxolanes (47), and tetrahydro- pyran derivatives (85)], and glycosides (82) it has been Shown that the more electron-attracting is the ring substitu- ent the more difficult is the hydrolysis of the acetal or the glycoside linkage. Again these results can best be exPlained by assuming that the hydrolysis proceeds via a cyclic carbonium ion (pathway A), rather than by an acyclic carbonium ion (pathway B) aS follows: 0% 2R 0% ,R H 0 0 0 0 Et fis ‘. + EtOH\‘ o o o o I/R 3/ \ affast Product 0 Et H0 0\\ R é w ‘3’ fast 0 H 0 Et The inductive effect of the ring amide, carboxyl, and ester substituents can produce a destabilization of the A 5 III 1 ‘7 III- I I | l 91 carbonium ion as well as a decrease in the ease of heteroly- SiS. .These two factors would decrease the hydrolysis rate constant, as observed. The rate-retarding effect of the amide, carboxyl, and ester substituents in the 6-ethoxy-2-substituted tetra- hydrOpyrans is, nevertheless, in accordance with the induc- tive constants of the substituents as Shown in Figure 7. This lack of carboxyl group participation is interesting since it has been suggested that a carboxyl group at the same relative position assists the hydrolysis of methyl-a—D— glucopyranosiduronic acid (86). It is posSible that the conformation of the 6-ethoxy— 2-substituted tetrahydroPyran is reSponSible for the ob- served lack of carboxyl group participation. ~A theoretical conformational analysis indicates that four conformational Species may be present in the compound being studied, two cis and two trans: COOH O 5 P Et (a) (b) COOH OEt COOH (c) (d) 92 The carboxylate group could participate in an intra- molecular nucleophilic displacement of the "aglycone" only in the (d) conformation, whereas the protonated carboxyl group could participate as a general acid only in the (b) conformation. Since none of this anchimeric assistance is actually observed, the straightforward conclusion is that either the carboxyl group does not participate or the actual conformation of the Species present in the compound does not allow such participation. From purely theoretical con- siderations it would appear that the (a) configuration, which apparently cannot allow carboxyl group participation, is the most abundant because of its conformational stability. That is, during the synthesis of the compound COOH COOH COOES \9 + +iEtOH + H+—) ‘. —>' Et + H + H20 the cis conformation with both substituents in the equatorial position could be favored. From the results obtained with the acetal and the three series of tetrahydropyran derivatives it can be concluded that these compounds undergo a Specific acid-catalyzed' hydrolysis. Their corresponding hydrolytic rate constants can be predicted from the 0 value of the reSpective subs stituent. 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