title: Effects of site-directed mutagenesis of the loop residue of the N-terminal domain Gly117 of thermolysin on its catalytic activity.
abstract: In the N-terminal domain of thermolysin, two polypeptide strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122, are connected by a short loop, Asn116-Gly117, to form an anti-parallel β-sheet. The Asn112-Trp115 strand is located in the active site, while the Ser118-Tyr122 strand and the Asn116-Gly117 loop are located outside the active site. In this study, we explored the catalytic role of Gly117 by site-directed mutagenesis. Five variants, G117A (Gly117 is replaced by Ala), G117D, G117E, G117K, and G117R, were produced by co-expressing in Escherichia coli the mature and pro domains as independent polypeptides. The production levels were in the order G117E > wild type > G117K, G117R > G117D. G117A was hardly produced. This result is in contrast to our previous one that all 72 active-site thermolysin variants were produced at the similar levels whether they retained activity or not (M. Kusano et al. J. Biochem., 145, 103-113 (2009)). G117E exhibited lower activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and higher activity in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester than the wild-type thermolysin. G117K and G117R exhibited considerably reduced activities. This suggests that Gly117 plays an important role in the activity and stability of thermolysin, presumably by affecting the geometries of the Asn112-Trp115 and Ser118-Tyr122 strands.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Evans
authors.initials: E
authors.lastname: Menach
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D000179:Acrylates / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis; D020134:Catalytic Domain; D002417:Cattle; D004151:Dipeptides / Q000378:metabolism; D004795:Enzyme Stability; D005998:Glycine; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000179:Acrylates; D004151:Dipeptides; C108649:N-(3-(2-furyl)acryloyl)glycyl-leucinamide; C051210:N-benzyloxycarbonyl-aspartylphenylalanine methyl ester; D013820:Thermolysin; D005998:Glycine