Journal of Biochemistry Advance Access originally published online on April 3, 2007
Journal of Biochemistry 2007 141(6):843-853; doi:10.1093/jb/mvm088
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© 2007 The Japanese Biochemical Society.
Kinetic Analysis of the Activation-and-Inhibition Dual Effects of Cobalt Ion on Thermolysin Activity
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 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 Co2+ ion on thermolysin activity in the hydrolysis of FAGLA was examined. Thermolysin activity increased 3–4 times with increasing the Co2+ concentration to 2 mM, but the enhanced activity was considerably reduced with higher Co2+ concentration (2–18 mM). The activation-and-inhibition dual effects of Co2+ ion were analysed kinetically. Release of the catalytic Zn2+ ion from thermolysin, concomitantly occurred with the Co2+-dependent activation, was measured with a Zn2+-specific fluorescent probe. This indicates that the activation is caused by substituting Co2+ ion for the catalytic Zn2+ ion. Meanwhile, the Co2+-dependent activation was inhibited competitively by Zn2+ ion (0.1–1.0 µM) added, similarly to that it is inhibited by higher concentration of Co2+ ion. These lines of evidence provide a strategy for regulating thermolysin activity with Co2+ and Zn2+ ions.
Key Words: cobalt ion, inhibition, metalloproteinase, thermolysin, zinc ion
Abbreviations: FA-, N-[3-(2-furyl)acryloyl]-; FAGLA, N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide; ZDFM, N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester; ZnAF-2, 6-[N-[N', N'-bis(2-pyridinylmethyl)-2-aminoethyl]amino-3',6'-dihydroxy-spiro [isobenzofuran-1(3H), 9'-[9H] xanthen]-3-one, tetrahydrochloride
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