Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO(-) group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibility of the transpeptidation reaction.     

Development of an HPLC assay for Staphylococcus aureus sortase: evidence for the formation of the kinetically competent acyl enzyme intermediate

Many bacterial surface proteins containing an LPXTG motif are anchored to the cell wall peptidoglycan by catalysis with the thiol transpeptidase sortase. The transpeptidation and hydrolysis reactions of sortase have been proposed to proceed through a common acyl enzyme intermediate. The reactions of Staphylococcus aureus sortase with fluorogenic substrate Abz-LPETG-Dnp in the presence or absence of triglycine were characterized in this study to gain additional insight into the kinetic mechanism of sortase. We report here the development of a reverse-phase HPLC assay to identify and characterize sortase reaction intermediates. The HPLC results provide for the first time clear evidence for the formation of a kinetically competent acyl enzyme intermediate during the overall transpeptidation reaction. The results also suggest that sortase undergoes an unexpected intramolecular acyl transfer reaction in the absence of a nucleophile. The significance of this type of HPLC assay as a tool to study enzyme mechanism is discussed.     

In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli

PBP1B is a major bifunctional murein (peptidoglycan) synthase catalyzing transglycosylation and transpeptidation reactions in Escherichia coli. PBP1B has been shown to form dimers in vivo. The K(D) value for PBP1B dimerization was determined by surface plasmon resonance. The effect of the dimerization of PBP1B on its activities was studied with a newly developed in vitro murein synthesis assay with radioactively labeled lipid II precursor as substrate. Under conditions at which PBP1B dimerizes, the enzyme synthesized murein with long glycan strands (>25 disaccharide units) and with almost 50% of the peptides being part of cross-links. PBP1B was also capable of synthesizing trimeric muropeptide structures. Tri-, tetra-, and pentapeptide compounds could serve as acceptors in the PBP1B-catalyzed transpeptidation reaction.     

Protein kinase activity in membrane preparations from ox brain. Stimulation of intrinsic activity by adenosine 3′:5′-cyclic monophosphate

When Ac(2)-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac(2)-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac(2)-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac(2)-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested.     

Kinetic studies of sheep kidney gamma-glutamyl transpeptidase.

The kinetics of sheep kidney gamma-glutamyl transpeptidase was studied using a novel substrate L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate. When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding L-alpha-methyl-gamma-glutamyl derivatives of the acceptors were formed. In the absence of acceptor only hydrolysis occurred, and no transpeptidation products were detected. The presence of the methyl group on the alpha-carbon apparently prevents enzymatic transfer of the L-alpha-methyl-gamma-glutamyl residue to the amino group of the substrate itself (autotranspeptidation). When the enzyme was incubated with conventional substrates, such as glutathione or gamma-glutamyl-p-nitroanilide and an amino acid acceptor, hydrolysis, autotranspeptidation, and transpeptidation to the acceptor occurred concurrently. Initial velocity measurements in which the concentration of L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate was varied at several fixed acceptor concentrations, and either the release of alpha-aminobutyrate or the formation of the transpeptidation products was determined, yielded results which are consistent with a ping-pong mechanism modified by a hydrolytic shunt. A scheme of such a mechanism is presented. This mechanism predicts the formation of an alpha-methyl-gamma-glutamyl-enzyme intermediate, which can react with an amino acid to form the transpeptidation product; or in the absence of, or in the presence of low concentrations of amino acids, can react with water to form the hydrolytic products. Kinetic derivations for the reaction of the enzyme with the conventional substrate gamma-glutamyl-p-nitroanilide predict either linear or nonlinear double-reciprocal plots, depending on the prevalence of the hydrolytic, autotranspeptidation, or transpeptidation reactions. The results of kinetic experiments confirmed these predictions.     

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.     

Modulation of gamma-glutamyl transpeptidase activity by bile acids

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.     

The effect of pH on the transpeptidation and hydrolytic reactions of rat kidney gamma-glutamyltransferase

The effect of pH upon the transpeptidation and hydrolytic reactions of gamma-glutamyltransferase [5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2) have been investigated. It was found that the enzyme was irreversibly inactivated below pH 7.5 or above pH 9.4. Transpeptidation was markedly pH-dependent, while hydrolysis was pH-independent. The pH optimum for transpeptidation was found to vary for different acceptors. The ascending limb of the pH-optimum curve is attributed to the pK of the alpha-amino group of the acceptor, while the descending limb of the pH-optimum curve is attributed to an ionisable group in the active site of the enzyme. These observations provide much information about the interaction of the enzyme with the acceptor: (1) the true acceptor for gamma-glutamyltransferase is the deprotonated form of the amino acid; (2) glycylglycine has a similar acceptor activity to methionine, its apparent higher activity being due to the low pK of the alpha-amino group; (3) the enzyme is reversibly inactivated at higher pH by the deprotonation of a group in the active site which is involved in both binding of acceptor and catalysis of transpeptidation (this group is not involved in the hydrolysis reaction); (4) at pH 8.5, the normal pH for assay, only 47% of the enzyme is active, while at pH 7.4 gamma-glutamyltransferase is 93% in the active form.     

In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors.     

The exchange reaction of peptides R-D-alanyl-D-alanine with D-[14C]alanine to R-D-alanyl-D-[14C]alanine and D-alanine, catalysed by the membranes of Streptococcus faecalis ATCC 9790.

Under alkaline conditions, the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC 9790 catalyses exchange reactions in which the X-L-R3-D-Ala moiety of peptides of the type X-L-R3-D-Ala-D-Ala is transferred to simple amino compounds such as D-alanine, glycine and glycyl-glycine. The enzyme system is unable, however, to catalyse complex reactions that would simulate the natural transpeptidation reaction.     

Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis

The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.     

Kinetic analysis of the action of tissue transglutaminase on peptide and protein substrates

Tissue transglutaminase (TGase) catalyzes transfer of gamma-acyl moieties of Gln residues in peptides or protein substrates to either water or amine nucleophiles through an acyl-enzyme intermediate formed from initial acyl-transfer to an active site Cys residue. Natural substrates for this enzyme include proteins (e.g., tau, alpha-synuclein, and huntingtin) whose TGase-promoted polymerization may be causative in neurodegenerative diseases. As part of a program to find inhibitors of TGase, we have undertaken kinetic and mechanistic studies of the enzyme from guinea pig (gpTGase) and humans (hTGase). Key findings of this study include: (i) gpTGase-catalyzed transamidation of Z-Gln-Gly by Gly-OMe proceeds essentially as described above but with the involvement of substrate inhibition by Gly-OMe. This phenomena, resulting from the binding of nucleophile to free enzyme, appears to be a common feature of TGase-catalyzed reactions. (ii) Solvent deuterium isotope effects for hydrolysis of Z-Gln-Gly by gpTGase are (D)(k(c)/K(m)) = 0.45 and (D)k(c) = 3.6. While the latter results from general catalysis of deacylation, the former originates purely from the reactant state, hydrogen fractionation factor of the active site thiol with no involvement of general catalysis of acylation. (iii) Studies of the transamidation of N,N-dimethylated casein by Gly-OMe and dansyl-cadaverine suggest a complex kinetic mechanism for both enzymes that reflects contributions from four reactions: Gln hydrolysis, intramolecular transpeptidation, intermolecular transpeptidation, and transamidation by added nucleophile.     

Acyl and amino intermediates in reactions catalyzed by thermolysin

Thermolysin showed peculiar transpeptidation reactions. Leu-Leu and/or Leu-Leu-Leu were produced at $\text{ca}$. pH 7 from Leu-Leu-NH₂ and Cbz-Leu-Leu. Isotope experiments indicated that the transpeptidation products did not use leucine released from the substrates as an acceptor. With Leu-Trp-Met, Leu-Leu, Leu-Leu-Leu and Met-Met were produced as transpeptidation products. A comparative study was done with α-chymotrypsin and pepsin. These results would indicate that thermolysin catalyzed reactions proceed via both acyl and amino intermediates depending upon the substrates, which has been proposed for the mechanism of pepsin. This may also be true in some cases for chymotrypsin and other proteases, which have been known as enzymes of the acyl-enzyme mechanism.     

Kinetic mechanism of Staphylococcus aureus sortase SrtA

Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis.     

Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney.

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.     

Identification and Characterization of a Gamma-Glutamyl Transpeptidase from a Thermo-Alcalophile Strain of Bacillus pumilus

A γ-glutamyltranspeptidase (GGT, E.C. 2.3.2.2) was isolated from a strain (A8) originating from Lake Bogoria (Kenya) and homologous with Bacillus pumilus. This GGT shows an optimal activity at pH 8.9 and 62°C. The enzyme is thermostable up to 43°C. The best reagent among the potential inhibitors was shown to be DON, which is an inhibitor highly specific for GGTs. Gly-Gly-Ala, Gly-Gly-Gly and Gly-Gly were identified as the best acceptors for the transpeptidation reactions catalyzed by the enzyme. The SDS-PAGE study revealed that the enzyme consists of two non-identical subunits (38,000 and 23,000). Only the large subunit was active when the enzyme was dissociated under denaturing conditions. The behavior of the native enzyme suggests that the active site of the large subunit is masked by the small subunit.     

Transpeptidation reactions of a specific substrate catalyzed by the Streptomyces R61 DD-peptidase: the structural basis of acyl acceptor specificity

Bacterial dd-peptidases, the targets of beta-lactam antibiotics, are believed to catalyze d-alanyl-d-alanine carboxypeptidase and transpeptidase reactions in vivo. To date, however, there have been few concerted attempts to explore the kinetic and thermodynamic specificities of the active sites of these enzymes. We have shown that the peptidoglycan-mimetic peptide, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, 1, is a very specific and reactive carboxypeptidase substrate of the Streptomyces R61 dd-peptidase [Anderson, J. W., and Pratt, R. F. (2000) Biochemistry 39, 12200-12209]. In the present paper, we explore the transpeptidation reactions of this substrate, where the enzyme catalyzes transfer of the glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl moiety to amines. These reactions are believed to occur through capture of an acyl-enzyme intermediate by amines rather than water. Experiments show that effective acyl acceptors require a carboxylate group and thus are amino acids and peptides. d(but not l)-amino acids, analogues of the leaving group of 1, are good acceptors. The effectiveness of d-alanine as an acceptor increases with pH, suggesting that the bound and reactive form of an amino acid acceptor is the free amine. Certain glycyl-l(but not d)-amino acids, such as glycyl-l-alanine and glycyl-l-phenylalanine, are also good acceptors. These molecules may resemble the N-terminus of the Streptomyces stem peptides that, presumably, are the acceptors in vivo. The acyl acceptor binding site therefore demonstrates a dual specificity. That d-alanyl-l-alanine shows little activity as an acceptor suggested that, on binding of acceptors to the enzyme, the carboxylate of d-amino acids does not overlap with the peptide carbonyl group of glycyl-l-amino acids. Molecular modeling of transpeptidation tetrahedral intermediates and products demonstrated the likely structural bases for the stereospecificity of the acceptors and the nature of the dual function acceptor binding site. For both groups of acceptors, the terminal carboxylate appeared to be anchored at the active site by interaction with Arg 285 and Thr 299.     

Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins.

Penicillopepsin catalyses transpeptidation reactions involving the transfer of the N-terminal amino acids of suitable substrates via covalent acyl intermediates to acceptor peptides, usually the substrate. The major products obtained when Phe-Tyr-Thr-Pro-Lys-Ala and Met-Leu-Gly were used as substrates were Phe-Phe and Met-Met respectively. With Met-Leu-Gly the tetrapeptide Met-Met-Leu-Gly was observed as probable intermediate. Co-incubation of Leu-Tyr-Leu and Phe-Tyr-Thr-Pro-Lys-Ala led to the formation of Leu-Phe and Phe-Leu as well as Leu-Leu and Phe-Phe. No reaction was observed with tripeptides in which the first or second amino acid is glycine. It appears that two amino aicds with large hydrophobic residues are needed for the transpeptidation reaction. Nucleophilic compounds other than peptides, such as hydroxylamine, aliphatic alcohols and dinitrophenylhydrazine, were not acceptors for the acyl group. Leucine, phenylalanine and leucine methyl ester also had no effect on the reaction. The transpeptidation reaction proceeded readily at pH 3.6 and 4.7. At pH 6.0 the reaction was slow and at pH 1.9 little or no transpeptidation was observed. Porcine pepsin catalyses similar transpeptidation reactions. Sequence studies show that porcine pepsin and penicillopepsin are homologous. The present study also suggests that they have a very similar mechanism. Evidence available at this time indicates that the mechanism of these enzymes is complex and may be modulated by secondary substrate-enzyme interactions. A hypothesis is presented which proposes that pepsin-catalysed reactions proceed via different covalent intermediates (amino-intermediates or acylintermediates) depending on the nature of the substrate. The possibility that some reactions do not involve covalent intermediates is also discussed.     

Carboxypeptidase Y stability

The stability of carboxypeptidase Y under different reaction conditions and in the presence various cosolvents was investigated. Loss of both hydrolysis and transpeptidation activities was monitored. Incubation of the enzyme at high temperatures or high pH resulted in the loss of both activities at the same rate. Addition of ammonium sulfate resulted in loss of transpeptidation activity but not hydrolysis activity. Addition of some organic solvents or Triton X-100 to the incubation mixture resulted in loss of both activities with transpeptidation being lost more rapidly than hydrolysis activity, while other organic solvents were observed to eliminate both activities entirely. Incubation of the enzyme in the presence of sodium dodecyl sulfate resulted in a decrease in both activities but hydrolysis was lost more rapidly than the transpeptidase activity. Implications of the observed preferential loss of activities to the labeling of peptides and proteins is discussed.     

A new strategy for the study of oligomeric enzymes: gamma-glutamyltransferase in reversed micelles of surfactants in organic solvents

A heterodimeric enzyme (gamma-glutamyltransferase) was studied in the reversed micellar medium of Aerosol OT (AOT) in octane. As was shown earlier, the size (radius) of inner cavity of the AOT-reversed micelles is determined by their hydration degree, i.e., [H2O]/[AOT] molar ratio, in the system. Owing to this, the dependence of hydrolytic, transpeptidation and autotranspeptidation activities of the enzyme on the hydration degree was investigated using L- and D-isomers of gamma-glutamyl(3-carboxy-4-nitro)anilide and glycylglycine as substrates. For all of the reaction types, the observed dependences are curves with three optima. The optima are found at the hydration degrees, [H2O]/[AOT] = 11, 17 and 26 when the inner cavity radii of reversed micelles are equal to the size of light (Mr 21,000) and heavy (Mr 54,000) subunits of gamma-glutamyltransferase, and to their dimer (Mr 75,000), respectively. Ultracentrifugation experiments showed that a change of the hydration degree resulted in a reversible dissociation of the enzyme to light and heavy subunits. The separation of light and heavy subunits of gamma-glutamyltransferase formed in reversed micelles was carried out and their catalytic properties were studied. The two subunits catalyze hydrolysis and transpeptidation reactions; autotranspeptidation reaction is detected only in the case of the heavy subunit. These findings imply that the reversed micelles of surfactants in organic solvents function as the matrices with adjustable size permitting to regulate the supramolecular structure and the catalytic activity of oligomeric enzymes.