Biochemical and molecular characterization of recombinant Bacillus subtilis tripeptidase (PepT) as a zinc-dependent metalloenzyme.
- Title
- Biochemical and molecular characterization of recombinant Bacillus subtilis tripeptidase (PepT) as a zinc-dependent metalloenzyme.
- Author
- 정일엽
- Issue Date
- 2000-08
- Publisher
- 한국분자생물학회
- Citation
- MOLECULES AND CELLS, v. 10, no. 4, Page. 423-431
- Abstract
- Aminopeptidases catalyze the release of N-terminal amino acid residue from polypeptides and peptides, and most of them are known
to be metalloenzymes. A tripeptidase gene (pepT) of Bacillus subtilis was expressed in Escherichia coli, and the resulting recombinant
PepT was purified in an active form through sequential chromatographies. The addition of Zn2+ or Co2+ increased the enzymatic
activity by approximately two fold. The points at which Zn2+ and Co2+ stimulated a half-maximum activity of the PepT were 650 nM
and 1,700 nM, respectively. The measurement of the metal content showed that this enzyme contained 0.26 atom of Zn2+ per
molecule with essentially the absence of Co2+ and others, and 0.53 atom of Zn2+ with 1.5-fold increase of activity when reconstituted
with Zn2+. Consistent with this result, this enzyme is much readily refolded in the presence of Zn2+ than Co2+. To further delineate
the structure and function relations, we made serial deletion mutants and analyzed their enzymatic activities. Of eight deletion mutants,
only a mutant lacking the N-terminal 66 amino acid residues retained enzymatic activity. The mutant enzyme, however, required a
concentration of Zn2+ ion at least ten-fold higher to reach maximum activity without significantly affecting kinetic parameters such as
Km and Vmax compared to the full length PepT. Taken together, these data suggest that the B. subtilis PepT is likely to be a
Zn2+-dependent metalloenzyme and that the N-terminal region of the PepT stabilizes Zn2+-binding.
- URI
- http://eds.a.ebscohost.com/eds/pdfviewer/pdfviewer?vid=1&sid=6645f83e-6970-40ad-b609-5b99c60b5c4a%40sdc-v-sessmgr02https://repository.hanyang.ac.kr/handle/20.500.11754/162870
- ISSN
- 1016-8478
- Appears in Collections:
- COLLEGE OF SCIENCE AND CONVERGENCE TECHNOLOGY[E](과학기술융합대학) > MOLECULAR AND LIFE SCIENCE(분자생명과학과) > Articles
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