酿酒酵母蛋白酶A的底物特异性和动力学特性以及一种新型底物的开发

Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate.

作者信息

Kondo H, Shibano Y, Amachi T, Cronin N, Oda K, Dunn B M

机构信息

Institute for Fundamental Research, Suntory Limited, Shimamoto-cho, Mishima-gun, Osaka, 618-0024, Japan.

出版信息

J Biochem. 1998 Jul;124(1):141-7. doi: 10.1093/oxfordjournals.jbchem.a022072.

DOI:10.1093/oxfordjournals.jbchem.a022072

PMID:9644256

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 (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-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.

摘要

利用一系列具有通用结构P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3'[P5、P4、P3、P2、P2'、P3'为各种氨基酸；(NO2)Phe为对硝基-L-苯丙氨酸]的合成生色肽，测定了来自酿酒酵母的细胞内天冬氨酸蛋白酶A的底物特异性和动力学性质。占据底物NH2末端区域的残基性质对动力学常数有很大影响。在测试的那些底物中，Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu具有最佳的动力学常数（Km = 0.012 mM，kcat = 14.4 s-1，kcat/Km = 1200 M-1·s-1）。与胃蛋白酶、组织蛋白酶D和肾素等天冬氨酸蛋白酶相比，蛋白酶A的底物特异性是独特的。基于这些结果，开发了一种新型荧光底物MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2，用于灵敏测定蛋白酶A。