Substrate Specificities and Kinetic Properties of Proteinase A from the Yeast Saccharomyces cerevisiae and the Development of a Novel Substrate

Abstract

The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO₂) Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO₂)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH₂-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO₂)-Phe-Arg-Leu had the best kinetic constants (K_m=0.012mM, k_cat=14.4 s^-1, k_cat/K_m=1, 200M^-1•s^-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH₂, was developed for the sensitive measurement of proteinase A.