The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis

Abstract

a-Chymotrypsin-catalyzed acyl tranfer was studied using three acyl-group donors (Mal-~-Ala-L-Ala-L-PheOMe, Bz-L-TyrOEt and Ac-L-TrpOEt ; Mal, maleyl; Bz, benzoyl ; OMe, methyl ester; OEt, ethyl ester) and a series of amino-acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl-enzyme. The a-chymotrypsin-catalyzed transfer of the Mal-L-Ah-L-Ala-L-Phe group to the amides of L-Phe and L-Tyr showed a linear dependence of the partition constant, p , on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl-enzyme-nucleophile complex. The a-chymotrypsincatalyzed transfer of the Bz-L-Tyr and A c -L -T~~ groups to several amino-acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl-enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of a-chymotrypsin-catalyzed acyl transfer evident in previous studies.In proteinase-catalyzed acyl-transfer reactions, the acyl group of a substrate can be transferred both to water and to the added nucleophile. As a result, the hydrolytic product donor, N is the acyl-group acceptor (added nucleophile, amino component), P is the peptide synthesized, P, and P, are the products of the hydrolysis of the acyl-group donor and ES is the enzyme complex with S and EA denoting the acyl-enzyme. The kinetic constants are indicated for the appropriate reactions in the scheme. where E represents the enzyme, S denotes the acyl-group

High level production of bioactive di- and tri-tyrosine peptides by protease-catalyzed reactions

“…The high specificity and high reactivity under mild conditions, which is characteristics of enzyme, can reduce operation steps required for protection-deprotection and avoid side reactions such as racemization. In the protease-catalyzed peptide synthesis, the acyl group of a substrate specific to the S subsite region in a protease is transferred to an amino component with a high affinity to the S subsite in the catalyst through the acylenzyme intermediate (Schuster et al, 1991;Gololobov et al, 1993;Lombard et al, 2005). This deacylation of the acyl-enzyme intermediate giving a peptide product is called aminolysis.…”

The 3D model of the lipase/acyltransferase from Candida parapsilosis, a tool for the elucidation of structural determinants in CAL-A lipase superfamily

“…Apart from those fungal lipases and an enzyme from Mycobacterium smegmatis reported to be able to catalyze alcoholysis in aqueous conditions [28], the great potential of lipases/ acyltransferases for the development of environmentally friendly enzymatic processes has been poorly discussed in lipase literature. Comparable "kinetically controlled" reactions have however been described for acyltransfer catalysis in water abundant media with other enzymes, such as for the synthesis of peptide bonds using proteases or for amineacylation catalyzed by penicillin-acylases [29][30][31][32][33][34][35][36][37][38][39][40]. Such biocatalysts also display intrinsic properties that determine a kinetically controlled synthesis with a superior transfer ratio compared to the corresponding "classical" hydrolases, which catalyze "equilibrium controlled" synthesis.…”

Chemoenzymatic Synthesis of Polypeptides for Use as Functional and Structural Materials

“…Trypsin selectively digests amide bonds next to basic amino acids, such as l-lysine and l-arginine, [39,40] www.advancedsciencenews.com www.mbs-journal.de α-chymotrypsin efficiently recognizes aromatic amino acids, such as l-phenylalanine and l-tyrosine. [41][42][43] Proteinase K, another serine protease, has high affinity for hydrophobic and aromatic amino acids [44] but can also polymerize hydrophilic amino acids, such as l-cysteine and l-histidine. [45] In contrast, among the cysteine proteases, papain is commonly used because it is an inexpensive and stable extracellular protease.…”