Yeast Dipeptidase: Active Site Mapping by Kinetic Studies with Substrates and Substrate Analogs

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Abstract
In order to characterize the active site of yeast dipeptidase [EC 3.4.13.?] in more detail, kinetic studies with a variety of dipeptide substrates and substrate analogs were performed. To analyze kinetic data, computer programs were developed which first calculate initial velocities from progress curves and then evaluate the kinetic parameters by nonlinear regression analysis. A free carboxyl group is a prerequisite for binding of dipeptidase substrates; its position relative to the peptide bond must not deviate from the normal L-dipeptide conformation. The spatial arrangement of the terminal ammonium ions seems to be less crucial. The enzyme''s substrate specificity clearly reflects the interactions of the substrate amino acid side chains with complementary dipeptidase subsites. The domain of the enzyme in contact with the C-terminal substrate side chain seems to be an open structure of moderately hydrophobic character. In contrast, the binding site for the amino-terminal side chain is a more strongly hydrophobic pocket of limited dimensions. The kinetics of inhibition by free amino acids points to an ordered release of products from the enzyme.

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