How to predict product yield in kinetically controlled enzymatic peptide synthesis.

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters alpha and beta for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH(2) from Mal-L-Phe-L-AlaOMe and L-LeuNH(2). The experimental results demonstrate that this theory adequately describes the real process.

Increased nucleophile reactivity of amino acid beta-naphthylamides in alpha-chymotrypsin-catalyzed peptide synthesis.

Alpha-chymotrypsin-catalyzed acyl transfer from Boc-L-MetONp, Ac-L-TyrOEt, Bz-L-TyrOMe, Mal-L-PheOMe to the C-protected amino acids (L-AlaNH2, L-LeuNH2, L-ArgOMe and beta-naphthylamides of L-Arg, L-Leu, L-Ala and L-Glu) has been studied. Modification of the carboxylic groups with beta-naphthylamide was shown to increase the reactivity of nucleophiles in these reactions by a factor of more than 100 in comparison with amides and esters of the same amino acids. This effect can be accounted for by the effective formation of the nucleophile-acylenzyme complex due to hydrophobic interactions of the beta-naphthylamide moiety with the corresponding subsite of alpha-chymotrypsin. The reaction kinetics follows the scheme involving hydrolysis of the nucleophile-acylenzyme intermediate. The contribution of this pathway depends on the structures of both the acyl-group donor and the added nucleophile. The competitive inhibition by amino acid beta-naphthylamides is also observed. The results obtained show that modification of the COOH-group of added nucleophiles by beta-naphthylamide strongly affects the reactivity of these compounds in the alpha-chymotrypsin-catalyzed peptide synthesis.

Acyl group transfer by proteases forming an acylenzyme intermediate: kinetic model analysis (including hydrolysis of acylenzyme- nucleophile complex).

The quantitative analysis of peptide synthesis via transfer of the acyl moiety from the activated donor (S) to the nucleophile (N), catalysed by proteases forming an acylenzyme intermediate, has been continued. The new kinetic model takes into account the hydrolysis of an acylenzyme-nucleophile complex (EAN). The intensity of the hydrolysis is characterized by parameter gamma equal to the ratio of the rate constant of EAN hydrolysis and the rate constant of peptide formation. The ability of the EAN complex to hydrolyse leads to a decrease in the apparent nucleophile reactivity (beta) of the aminocomponent. As a result, the maximal fractional conversions of S and N to the peptide decrease, and the apparent nucleophile reactivity becomes dependent on the nucleophile concentration. The pattern of parameter gamma influence on maximal fractional conversions depends on which component is in an excess. It is with the donor excess that hydrolysis of the EAN complex affects the peptide yield dramatically. Analytical expressions for the estimation of maximal product concentration were obtained and their accuracy evaluated.

Acyl group transfer by proteases forming acyl-enzyme intermediate: kinetic model analysis.

A kinetic model of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate has been analyzed. The kinetic model takes into account the subsequent enzymatic hydrolysis of synthesized peptide (P), and so the kinetic curve for this compound shows a maximum (denoted as p(max)). Particular stress is placed on analyzing the effects of initial concentrations and of kinetic constants on the value of p(max).The analysis has demonstrated that at a given ratio of initial S and N concentrations, p(max) is affected only by (i) the ratio of the second-order rate constants for enzymatic hydrolysis of S and P(alpha) and (ii) the ratio of rate constants for an attack of the acyl-enzyme intermediate by the nucleophile and water (beta). These conclusions apply regardless of the existence of linear inhibition by the components of the reaction mixture. Thus, the kinetically controlled maximum yield of peptide (p(max)) can be calculated a priori from values of alpha and beta that can be estimated experimentally or from reference data. Simple analytical expressions were obtained, allowing a fairly accurate prediction of p(max) for a broad spectrum of S and N initial concentrations.

[Peptide synthesis catalyzed by proteases. Analysis of a kinetic model for enzymes with acyl-enzyme mechanism of action]. / Peptidnyi sintez, kataliziruemyi proteazami. Analiz kineticheskoi modeli dlia fermentov, deistvuiushchikh po atsilfermentnomu mekhanizmu.

The kinetics of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids or peptides (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate, was analysed. A kinetic model assumes enzymatic hydrolysis of the formed peptide (P), so the kinetic curve for P has a maximum (denoted as pmax). Particular attention was given to the analysis of the effects of the initial concentrations and kinetic constants on pmax. Computer analysis demonstrated that at a given ratio of initial S and N concentrations pmax is affected only by the ratio of the second order rate constants for enzymatic hydrolysis of S and P (alpha) and the ratio of rate constants for an attack of the acyl-enzyme intermediate by nucleophile and water (beta). These conclusions apply regardless of the existence of enzyme forms other than a free enzyme and an acyl-enzyme intermediate. Thus, the kinetically controlled maximum yield of peptide (pmax) can be calculated a priori from the values of alpha and beta which can be readily evaluated from the reference data. Simple explicit expressions were obtained, allowing fairly accurate prediction of pmax for a broad spectrum of S and N initial concentrations.