Effects of site-directed mutagenesis of Asn116 in the β-hairpin of the N-terminal domain of thermolysin on its activity and stability.
In the N-terminal domain of thermolysin, two anti-parallel β-strands, Asn112-Ala113-Phe114-Trp115 and Ser118-Gln119-Met120-Val121-Tyr122 are connected by an Asn116-Gly117 turn to form a β-hairpin structure. In this study, we examined the role of Asn116 in the activity and stability of thermolysin by site-directed mutagenesis. Of the 19 Asn116 variants, four (N116A, N116D, N116T and N116Q) were produced in Escherichia coli, by co-expressing the mature and pro domains separately, while the other 15 were not. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) at 25°C, the intrinsic k(cat)/K(m) value of N116D was 320% of that of the wild-type thermolysin (WT), and in the hydrolysis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM) at pH 7.5 at 25°C, the k(cat)/K(m) value of N116D was 140% of that of WT, indicating that N116D exhibited higher activity than WT. N116Q exhibited similar activity as WT, and N116A and N116T exhibited reduced activities. The first-order rate constants, k(obs), of the thermal inactivation at 80°C were in the order N116A, N116D, N116T > N116Q > WT at all CaCl(2) concentrations examined (1-100 mM), indicating that all variants exhibited reduced stabilities. These results suggest that Asn116 plays an important role in the activity and stability of thermolysin presumably by stabilizing this β-hairpin structure.
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Evans
E
Menach
Kiyoshi
K
Yasukawa
Kuniyo
K
Inouye
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics*; D002417:Cattle; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D050505:Mutant Proteins; D001216:Asparagine; D013820:Thermolysin