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Journal of Biochemistry Advance Access originally published online on October 30, 2008
Journal of Biochemistry 2009 145(1):103-113; doi:10.1093/jb/mvn140
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Insights into the Catalytic Roles of the Polypeptide Regions in the Active Site of Thermolysin and Generation of the Thermolysin Variants with High Activity and Stability

Masayuki Kusano, Kiyoshi Yasukawa and Kuniyo Inouye*

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 September 6, 2008; Accepted October 22, 2008


  Abstract

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112–Trp115), {alpha}-helix 1 (Val139–Thr149), C-terminal loop 1 (Asp150–Gly162), {alpha}-helix 2 (Ala163–Val176) and C-terminal loop 2 (Gln225–Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and {alpha}-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the kcat/Km values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2–3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80°C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.

Key Words: active site, metalloproteinase, site-directed mutagenesis, stability, thermolysin

Abbreviations: FAGLA, N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide; ZDFM, N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester


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