Inouye K, Lee S B, Tonomura B
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Biochem J. 1996 Apr 1;315 ( Pt 1)(Pt 1):133-8. doi: 10.1042/bj3150133.
The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced in the presence of high concentrations (1-5 M) of neutral salts [Inouye (1992) J. Biochem. (Tokyo) 112, 335-340]. In this study, the effect of salts on such activity has been examined using a series of substrates, furylacryloyl dipeptide amides, which have various hydrophobic amino acids at the cleavable bond. Although the enzyme activity varies widely depending on the substrate employed, the degree of activation at a given concentration of NaCl is considerably similar. This indicates that the degree of activation is not dependent on the hydrophobicity of the amino acid side chains at the scissile bond of the substrates. The molecular activity, kcat, and Michaelis constant, Km, were evaluated separately for substrates N[3-(2-furyl)acryloyl]-L-leucyl-L-alanine amide and N-[3-(2-furyl)acryloyl]L-phenyl-alanyl-L-alanine amide, and the activation was found to be brought about only by an increase in k(cat'). The effectiveness of monovalent cations on the increase of k(cat) was determined to follow the order of Na(+)>K(+)>Li(+).
在高浓度（1 - 5 M）中性盐存在下，嗜热菌蛋白酶对N - [3 - （2 - 呋喃基）丙烯酰基] - 甘氨酰 - L - 亮氨酸酰胺和N - 苄氧羰基 - L - 天冬氨酰 - L - 苯丙氨酸甲酯的水解活性显著增强[猪野（1992）《生物化学杂志》（东京）112，335 - 340]。在本研究中，使用了一系列在可裂解键处具有不同疏水氨基酸的底物——呋喃丙烯酰二肽酰胺，来检测盐对这种活性的影响。尽管酶活性因所使用的底物不同而有很大差异，但在给定浓度的氯化钠下的激活程度相当相似。这表明激活程度不依赖于底物可裂解键处氨基酸侧链的疏水性。分别对底物N - [3 - （2 - 呋喃基）丙烯酰基] - L - 亮氨酰 - L - 丙氨酸酰胺和N - [3 - （2 - 呋喃基）丙烯酰基] - L - 苯丙氨酰 - L - 丙氨酸酰胺评估了分子活性kcat和米氏常数Km，发现激活仅由k(cat')的增加引起。单价阳离子对k(cat)增加的有效性按Na(+)>K(+)>Li(+)的顺序排列。
