Journal of Biochemistry Advance Access originally published online on April 3, 2007
Journal of Biochemistry 2007 141(6):879-888; doi:10.1093/jb/mvm089
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© 2007 The Japanese Biochemical Society.
Molecular Mechanism of the Inhibitory Effect of Cobalt Ion on Thermolysin Activity and the Suppressive Effect of Calcium Ion on the Cobalt Ion-dependent Inactivation of Thermolysin
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
*To whom correspondence should be addressed. Tel: +81-75-753-6266, Fax: +81-75-753-6265, E-mail: inouye{at}kais.kyoto-u.ac.jp
Received February 10, 2007; Accepted March 28, 2007
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Abstract |
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Thermolysin activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) and FA-L-leucyl-L-alanine amide (FALAA) was examined at various Co2+ and Ca2+ concentrations. It decreased to 28% with increasing [Co2+] up to 18 mM. The Co2+-dependent inactivation was in part suppressed by adding Ca2+ ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca2+ (>0.5 mM). The Co2+-dependent inactivation was shown to be composed of Ca2+-sensitive and Ca2+-insensitive parts. In the latter part which is observed at [Ca2+] >0.5 mM, Co2+ plays as a competitive inhibitor. On the other hand, the Co2+-dependent inactivation in the Ca2+-sensitive part observed at [Ca2+] <0.5 mM proceeds time-dependently following second-order kinetics, and the time-course is in good agreement with that of decrease in the thermolysin band due to autolysis in SDS–PAGE. This indicates that Co2+ accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co2+ and Ca2+ ions and propose a molecular mechanism for the inhibition of thermolysin by Co2+ and suppressive effect of Ca2+ on the Co2+-dependent inhibition. Co2+ ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis.
Key Words: autolysis, cobalt, inhibition, metalloproteinase, thermolysin
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