Malt carboxypeptidase catalyzed aminolysis reactions

Abstract

Malt carboxypeptidase catalyzes the formation of peptide bonds using N-benzoyl-arginine esters as acyl components and amino acid amides or amino acid methyl esters as nucleophiles. With seven different nucleophiles yields of 50-95% were obtained while no aminolysis was observed with H-Pro-NH2 and H-GIu-a-NH2. The enzyme is easily saturated with nucleophile indicating the formation of a complex between nucleophile and acyl-enzyme intermediate prior to deacylation. The nature of the side-chain of the amino acid esters used as nucleophiles strongly influences the apparent dissociation constant of this complex (KN~app~) while less dependence on the side-chain is observed with amino acid amides. KN~app~ is drastically increased by the presence ofphenylguanidine in the reaction medium suggesting a competition between this substance and the nucleophile for the same binding site. This site has previously been identified as the S~ binding site of malt carboxypeptidase (Carlsberg Res. Commun. 48,573-582 (1983)). The influence of the nucleophile H-VaI-NH2 on the kinetic parameters for the cleavage of N,-CBZ-Lys-p-nitrophenylesler indicates that the acylation step is rate-limiting, and that H-Val-NH2 has two different modes of binding to the enzyme.

Serine carboxypeptidases. A review

“…The structures of the acyl and amine components which may participate in the reaction are determined by the specificity of the enzyme used, and studies in this laboratory have revealed that serine carboxypeptidases, e.g. carboxypeptidase Y and the malt carboxypeptidases, as expected, catalyze only aminolysis reactions with amino acids and amino acid derivatives as amine components (33,38,196,197) as opposed to endopeptidases that in addition to amino acid derivatives also accept polypeptides as nucleophiles (136,149). Consequently, when a carboxypeptidase is used a peptide chain will only be elongated by a single amino acid residue in each aminolysis reaction.…”

“…However, the limited data must be evaluated with caution since the situation where k2,~ k3 cannot be distinguished from k2 k3 as long as only the steady-state kinetics of hydrolysis reactions have been studied. Determination of kinetic parameters in the presence of added nucleophiles is more informative and such studies have been performed with malt carboxypeptidase I in this laboratory using ZLys 89 as substrate and H-Val-NH2 as added nucleophile (33). Under these conditions the acyl-enzyme intermediate is partitioned between hydrolysis and aminolysis products, i.e.…”

“…to alcohols, amino acids and amino acid derivatives (24,33,38,195,196,197). For carboxypeptidase Y it has been shown, using N-blocked amino acid esters as substrates, that the fraction ofaminolysis, P3/(P2+P3), is highly dependent on the nature of the amine nucleophile added.…”

“…the S~ binding site. By studying the influence of H-VaI-NH2 on the steady-state parameters, k~,, and Km, for the action of malt carboxypeptidase on N~t-CBZLys-ONp it was demonstrated that this enzyme also has two binding modes for the added nucleophile, of which only one causes aminolysis of the acyl-enzyme intermediate (33). More recently RIECHMANN and KASCHE have found that nucleophiles similarly bind to chymotrypsin prior to their participation in the deacylation reaction (156), suggesting that such binding is general for proteolytic enzymes.…”

Isolation of carboxypeptidase III from malted barley by affinity chromatography

“…The serine carboxypeptidases play an important role in this process and MmOLA ( 17,18,19) has presented evidence for the presence in malt of five such enzymes with complementary specificites. In this laboratory two of these have been isolated by affinity chromatography and their enzymatic properties and sequences have been determined (2,3,4,6,7,23,24). The isolation and characterization of CPD-Mm is reported here.…”