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.
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 Co(2+) and Ca(2+) concentrations. It decreased to 28% with increasing [Co(2+)] up to 18 mM. The Co(2+)-dependent inactivation was in part suppressed by adding Ca(2+) ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca(2+) (>0.5 mM). The Co(2+)-dependent inactivation was shown to be composed of Ca(2+)-sensitive and Ca(2+)-insensitive parts. In the latter part which is observed at [Ca(2+)] >0.5 mM, Co(2+) plays as a competitive inhibitor. On the other hand, the Co(2+)-dependent inactivation in the Ca(2+)-sensitive part observed at [Ca(2+)] <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 Co(2+) accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co(2+) and Ca(2+) ions and propose a molecular mechanism for the inhibition of thermolysin by Co(2+) and suppressive effect of Ca(2+) on the Co(2+)-dependent inhibition. Co(2+) ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis.
Hashida
Yasuhiko
Y
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Inouye
Kuniyo
K
D000179:Acrylates / Q000494:pharmacology; D001329:Autolysis; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008954:Models, Biological; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D000179:Acrylates; D004151:Dipeptides; D007477:Ions; C108649:N-(3-(2-furyl)acryloyl)glycyl-leucinamide; D003035:Cobalt; D013820:Thermolysin; C018021:cobaltous chloride; D002118:Calcium