Mechanism of carboxypeptidase‐Y‐catalysed peptide semisynthesis

Abstract

The initial rate steady-state kinetics of carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions with some a-N-benzoyl-L-tyrosinyl compounds has been investigated using L-valinamide as the nucleophile in aminolysis. Hydrolysis of a-N-benzoyl-L-tyrosinyl ethyl ester, 4-nitroanilide, and -amide has been studied in the pH range 4 -9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate-determining with the ester substrate, but with the 4-nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate-determining step is apparent in the range pH 4-9. For the 4-nitroanilide and the amide substrates, the kinetic parameter, k,/K,, is influenced by an ionizing group with a pK value of 6. Probably this is the active-site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate.Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here Lvalinamide was used as model nucleophile. The analysis of the observed kinetic eflects of L-valinamide on the initial rate behaviour of carboxypeptidase-Y-catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl-enzyme complex forming an enzyme-aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined.The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrate. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their kJKm values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.Carboxypeptidase Y from baker's yeast belongs to the group of serine carboxypeptidases. These enzymes are functionally similar, but structurally unrelated to the serine endopeptidases (Dreddam, 1986;Liao and Remington, 1990). Carboxypeptidase Y catalyzes the hydrolysis of peptide and ester bonds and exhibits broad substrate specificity (Bai et al., 1975;Hayashi et al., 1975).Recently, the enzyme has been successfully used in enzymic peptide synthesis (Widmer and Johansen, 1979;Hellio et al., 1988). The hydrolysis of substrates apparently proceeds via the classical serine protease mechanism (Quellet and Stewart, 1959) which involves enzyme-substrate complex formation followed by acylation and deacylation of the...

Site-directed mutagenesis on (serine) carboxypeptidase Y. A hydrogen bond network stabilizes the transition state by interaction with the C-terminal carboxylate group of the substrate

“…Two possibilities exist: hydrolysis at low pH may take place by a mechanism different from that of the serine endopeptidases, or alternatively, the p of His397 is below 3.0 and is not the 4.47 value on which kQÍt is dependent in the pH range investigated in this study. The pH profile of fccat for the hydrolysis of the ester substrate Bz-Tyr-OEt (Christensen et al, 1992) is similar to that reported here for a peptide substrate, and consequently, it is unlikely that the unusual shape of the pH profile is determined by the carboxylate group of the peptide substrate. With the double mutant N51A+E145A the pH profile for Arcat yields a pA"a identical to the value obtained with the wild-type enzyme, demonstrating that the ionization of the residue influencing fccat is not perturbed by the residues involved in binding of the C-terminal carboxylate group.…”

γ-Glutamyltranspeptidase-catalysed acyl-transfer to the added acceptor does not proceed via the ping-pong mechanism

“…a-Chymotrypsin-catalysed acyl-transfer and, in general, acyl-transfer reactions catalysed by serine proteases proceed without formation of the ESN complex (Scheme 3). P1 p Scheme 3 Scheme 3 was made using the results of this work and the previously referred studies of a-chymotrypsin, as well as previous studies of trypsin, carboxypeptidases Y and W and alkaline mesentericopeptidase (Seydoux et al, 1969;Riechman andKasche, 1984, 1985;Shima et al, 1987;Bratovanova et al, 1988;Christensen et al, 1992).…”

Synthesis of peptides with narrow molecular weight distributions via exopeptidase-catalyzed aminolysis of hydrophobic amino-acid alkyl esters

“…The increased size of the ester group may have hindered the optimal binding of the amino-acid esters to CPDY, and as a result, hydrolysis dominated. 23,32 The yields of the oligo(L-leucine), oligo(L-isoleucine) and oligo(L-valine) peptides were 76.6%, 81.3% and 77.9%, respectively, indicating that a small change in the chemical structure of the amino-acid side chains did not influence the aminolysis yields. In contrast, the molecular weights of the oligo(L-leucine), oligo(L-isoleucine) and oligo(L-valine) peptides were 612, 680 and 620 Da, respectively.…”

“…A kinetic model for serine protease-catalyzed peptide synthesis has been proposed. 19,[22][23][24][25][26] This model suggests that the protease-catalyzed synthesis of peptides proceeds via kinetically controlled synthesis. The activated amino acid and the enzyme first form an acyl-enzyme intermediate (ES complex) (Scheme 1), which is then competitively deacylated by a nucleophile or water.…”