Effects of site-directed mutagenesis of Asn116 in the  -hairpin of the N-terminal domain of thermolysin on its activity and stability

Abstract

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.

Effects of Mutations of Thermolysin, Asn116 to Asp and Asp150 to Glu, on Salt-Induced Activation and Stabilization

“…Of the 19 variants of Asn116, only N116D exhibited higher activity than WT while maintaining a high level of NaCl-induced activation, comparable to that of WT. 19) This is unique in comparison to our earlier work, wherein variants with higher activity than WT, such as D150E, exhibited drastically reduced degrees of NaCl-induced activation. 17) In this study, to explore the mechanism of the saltinduced activation and stabilization of thermolysin, we compared the effects of Asn116!Asp and Asp150!…”

“…In the absence of NaCl, the activities of N116D and D150E are about 3 times higher than that of wild-type thermolysin (WT). 17,19) In the absence of NaCl, Asn116!Asp decreased stability, while Asp150!Glu did not change it. Asn116 is located in the -hairpin structure in the N-terminal domain, and Asp150 is located in the active-site loop in the Cterminal domain (Fig.…”

“…12) We have identified several mutations that increase the hydrolytic activity for FAGLA and ZDFM. [16][17][18][19] Of such mutations, Asn116!Asp 19) (here the mutation of Asn116 to Asp is designated as Asn116!Asp, and the thermolysin variant bearing the mutation of Asn116! Asp as N116D) and Asp150!Glu 17) exhibited the strongest effects.…”

“…The thermal inactivation of thermolysin was examined by methods described previously. 19) Briefly, 200 mL of a solution containing 2.0 mM thermolysin, 40 mM HEPESNaOH, 10 mM CaCl 2 , and 0, 0.5, 1.0, 2.0, 2.5, 3.0, or 4.0 M NaCl pH 7.5 was incubated at 70 C for specified durations (6, 12, 18, 24, or 30 min). Then it was incubated at 25 C for 6 min.…”

Effects of Conversion of the Zinc-Binding Motif Sequence of Thermolysin, HEXXH, to That of Dipeptidyl Peptidase III, HEXXXH, on the Activity and Stability of Thermolysin

“…The casein-hydrolyzing activities of the thermolysin variants were measured by methods described previously. 19,23) Briefly, a thermolysin solution (0.5 mL) containing the soluble fractions corresponding to 5 mL of culture medium were added to 1.5 mL of a solution containing 1.33% (w/v) casein and 40 mM TrisHCl (pH 7.5) and incubated at 25 C for 30 min. The reaction was stopped by the addition of 2 mL of a solution containing 0.11 M trichloroacetic acid, 0.22 M sodium acetate, and 0.33 M acetic acid.…”