Kinetic analysis of the activation-and-inhibition dual effects of cobalt ion on thermolysin activity.

Thermolysin activity as well as its stability is remarkably enhanced by high concentration of neutral salts consisting of Na(+), K(+), Cl(-) and Br(-) in the synthesis and hydrolysis of N-carbobenzoxy-L-aspertyl-L-phenylalanine methyl ester and hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) [Inouye, K. (1992) J. Biochem. 112, 335-340]. However, effect of divalent salts on thermolysin activity has not been investigated systematically. In this study, effect of Co(2+) ion on thermolysin activity in the hydrolysis of FAGLA was examined. Thermolysin activity increased 3-4 times with increasing the Co(2+) concentration to 2 mM, but the enhanced activity was considerably reduced with higher Co(2+) concentration (2-18 mM). The activation-and-inhibition dual effects of Co(2+) ion were analysed kinetically. Release of the catalytic Zn(2+) ion from thermolysin, concomitantly occurred with the Co(2+)-dependent activation, was measured with a Zn(2+)-specific fluorescent probe. This indicates that the activation is caused by substituting Co(2+) ion for the catalytic Zn(2+) ion. Meanwhile, the Co(2+)-dependent activation was inhibited competitively by Zn(2+) ion (0.1-1.0 muM) added, similarly to that it is inhibited by higher concentration of Co(2+) ion. These lines of evidence provide a strategy for regulating thermolysin activity with Co(2+) and Zn(2+) ions.