A kinetic study of hydrophobic interactions at the S1 and S2 sites of papain

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Abstract
Two series of peptide substrates of papain, N-benzyloxycarbonyl-Y-X-OCH3 and N-benzyloxycarbonyl-L-alanyl-L-phenylalanyl-X-OCH3 in which X and Y represent L-alanine, L-leucine, and L-phenylalanine, were synthesized. All hydrolyze at the ester linkage in the presence of papain. The kinetic parameters kcat and Km were determined and interpreted in terms of binding interactions at the S1 and S2 sites of the enzyme. Significant kinetic specificity is seen in k3 (= kcat) as well as in kcat/Km. At the S1 site, deacylation reflects a preference for L-alanine by factors of 4- to 9-fold over L-leucine and 10-fold over L-phenylalanine provided the substrate has L-phenylalanine in the P2 position. There is no significant specificity at the S1 site reflected in the parameter kcat/Km. At the S2 site the well-known preference for L-phenylalanine is always more pronounced in the parameter kcat/Km than in k3. Indeed, deacylation is sometimes slower when L-phenylalanine rather than L-alanine or L-leucine interacts at the S2 site. A possible correlation between the effects of amino acid replacements at the S1 site on deacylation rates for substrates and inhibition constants for peptide inhibitors is discussed. Five dipeptide amide substrates were synthesized and their kinetic parameters were determined at pH 8 and pH 9. All hydrolyze at the amide linkage except N-benzyloxycarbonyl-L-alanyl-L-phenylalanine amide which hydrolyzes at the peptide linkage (55%) and the amide linkage (45%). Each of three amide substrates has nearly identical acylation and deacylation rates which may be a sign that the active site of papain has attained evolutionary perfection. The results are discussed in terms of the three-dimensional structure of papain.