Protease-catalyzed peptide bond formation: application to synthesis of the COOH-terminal octapeptide of cholecystokinin.

Abstract

This study of protease-catalyzed peptide synthesis reports the preparation of the COOH-terminal octapeptide amide of cholecystokinin. The octapeptide was assembled by chemical condensation oftwo tetrapeptide segments that had been synthesized through the concerted catalytic reactions of several proteases of different specificities. The resulting octapeptide derivative was subjected to catalytic transfer hydrogenation, followed by sulfation of its tyrosine residue and removal of the N'-protecting group. The homogeneous target peptide was obtained after purification via partition chromatography, gel filtration, and ion-exchange chromatography. The synthetic octapeptide stimulated amylase release from pancreatic acinar cells.

Subtiligase-Catalyzed Peptide Ligation

“…An alternative approach for using proteases in reverse is to run the reaction under kinetically controlled conditions, in which the kinetic properties of the enzyme (Δ G ‡ ), rather than the thermodynamic stabilities of the substrates and products, determine the reaction yield (Figure B). , This approach has been applied in the context of serine and cysteine proteases, − whose reaction mechanisms involve the formation of an acyl-enzyme intermediate. , In the context of these enzymes, the protease-catalyzed hydrolysis of both esters and amides proceeds through a similar chemical mechanism. The catalytic nucleophile (Ser or Cys) attacks the carbonyl bond of the substrate to form the acyl-enzyme intermediate.…”

Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin

“…This discrepancy may be rationalized if one bears in mind that in chymotrypsin-catalysis, in contrast with papain-catalysis, the acyl donors were esterified. As pointed out by Oka & Morihara (1978) for chymotrypsin, and more recently for papain by Doring et al (1981) and Kullmann (1982), ester donors are more efficient for enzymic peptide synthesis than are their corresponding free acids. Thus in the present study the catalytic constant for the papain-mediated synthesis of BocGly-Phe-N2H2Ph could be increased by roughly one order of magnitude by replacing Boc-Gly by Boc-Gly-OEt under otherwise comparable conditions.…”

Successful Application of the Lipase as a Catalyst in the Combinative Approach, Using an Efficient and Selective Iterative Procedure for the Chemo-Enzymatic Alpha-Amino Group Protection in the Natural Amino Acids, Representing the Ornithyl Model System as a Starting Research Pattern: Homogeneous and Heterogeneous Synthesis

“…This allows for the shifting of the reaction equilibrium towards the formation of the product. Usually, the precipitated product is collected by filtration of the reaction medium, which consists besides the product and the enzyme, also the unreacted excess from one of the components, which usually discards [15][16][17][18][19]. This methodology suffers from many disadvantages, due in many cases to the expensive reagents (costliness of the acyl or amino components), composing the related"pool" which means, that enzyme synthesis cannot be carried out in the presence of the observed low nucleophilic or electrophilic specificity, that would require the presence of a large excess from one, or from the another of the components (nucleophilic (amino) or electrophilic (acyl) compounds), which after remains unused.…”