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
| Autor: | Kuniyo Inouye, Evans Menach, Kiyoshi Yasukawa, Yasuhiko Hashida |
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| Rok vydání: | 2013 |
| Předmět: |
Models
Molecular Amino Acid Motifs Thermolysin chemistry.chemical_element Zinc Substrate analog Protein Engineering medicine.disease_cause Applied Microbiology and Biotechnology Biochemistry Analytical Chemistry Structure-Activity Relationship chemistry.chemical_compound Enzyme Stability medicine Animals Structure–activity relationship Dipeptidyl-Peptidases and Tripeptidyl-Peptidases Molecular Biology Escherichia coli Histidine Metalloproteinase Organic Chemistry Mutagenesis Cobalt General Medicine chemistry Mutagenesis Site-Directed Cattle Biotechnology |
| Zdroj: | Bioscience, Biotechnology, and Biochemistry. 77:1901-1906 |
| ISSN: | 1347-6947 0916-8451 |
| DOI: | 10.1271/bbb.130360 |
| Popis: | Most zinc metalloproteinases have the consensus zinc-binding motif sequence HEXXH, in which two histidine residues chelate a catalytic zinc ion. The zinc-binding motif sequence of thermolysin, H(142)ELTH(146), belongs to this motif sequence, while that of dipeptidyl peptidase III (DPP III), H(450)ELLGH(455), belongs to the motif sequence HEXXXH. In this study, we examined effects of conversion of HEXXH to HEXXXH in thermolysin on its activity and stability. Thermolysin variants bearing H(142)ELLGH(146) or H(142)ELTGH(146) (designated T145LG and T145TG respectively) were constructed by site-directed mutagenesis and were produced in Escherichia coli cells by co-expressing the mature and pro domains separately. They did not exhibit hydrolyzing activity for casein or N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but exhibited binding ability to a substrate analog glycyl-D-phenylalanine (Gly-D-Phe). The apparent denaturing temperatures based on the ellipticity at 222 nm of T145LG and T145TG were 85 ± 1 °C and 86 ± 1 °C respectively, almost the same as that of wild-type thermolysin (85 ± 1 °C). These results indicate that conversion of HEXXH to HEXXXH abolishes thermolysin activity, but does not affect its binding ability to Gly-D-Phe or its stability. Our results are in contrast to ones reported previously, that DPP III variants bearing H(450)ELGH(455) exhibit activity. |
| Databáze: | OpenAIRE |
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