The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Effects of Cobalt-Substitution of the Active Zinc Ion in Thermolysin on _??_ts Activity and Active-Site Microenvironment
Keiko KuzuyaKuniyo Inouye
Author information
JOURNAL FREE ACCESS

2001 Volume 130 Issue 6 Pages 783-788

Details
Abstract

Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4 M NaCI at pH 7.0 and 25°C Substitution of the active site zinc with other transition metals alters the activity of thermolysin [Holmquist, B. and Vallee, B. L. (1974) J. Biol. Chem. 249, 4601-4607]. Cobalt is the most effective among the transition metals and doubles the activity toward N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide. In this study, the effect of NaCI on the activity of cobalt-substituted thermolysin was examined. Cobalt-substituted thermolysin, with 2.8-fold increased activity compared with the native enzyme, is further activated by the addition of NaCl in an exponential fashion, and the activity is enhanced 13-15 times at 4M NaCl. The effects of cobalt-substitution and the addition of salt are independent of each other. The activity of cobalt-substituted thermolysin, expressed as kcat/Km, is pH-dependent and controlled by at least two ionizing residues with pKa, values of 6.0 and 7.8, the acidic pKa, being slightly higher compared to 5.6 of the native enzyme. These pKa, values remain constant in the presence of 4 M NaCl, indicating that the electrostatic environment of cobaltsubstituted thermolysin is more stable than that of the native enzyme, the acidic pKa, of which shifts remarkably from 5.6 to 6.7 at 4M NaCl. Zincov, a competitive inhibitor, binds more tightly to the cobalt-substituted than to native thermolysin at pH 4.9-9.0, probably because of its preference for cobalt in the fivefold coordination. The cobalt substitution has been shown to be a favorable tool with which to explore the active-site microenvironment of thermolysin.

Content from these authors
© The Japanese Biochemical Society
Previous article Next article
feedback
Top