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.
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; D001665:Binding Sites; D001966:Bromine / Q000737:chemistry; D002384:Catalysis; D002712:Chlorides / Q000494:pharmacology; D002713:Chlorine / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry*; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D011188:Potassium / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D013820:Thermolysin / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry; D017967:Zinc Compounds / Q000494:pharmacology
D000179:Acrylates; D002712:Chlorides; D004151:Dipeptides; D007477:Ions; C108649:N-(3-(2-furyl)acryloyl)glycyl-leucinamide; D017967:Zinc Compounds; D003035:Cobalt; D002713:Chlorine; C016837:zinc chloride; D012964:Sodium; D013820:Thermolysin; D015032:Zinc; D011188:Potassium; D001966:Bromine