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Metallo-β-lactamase : Structural basis for substrate-specificity and rational design of the inhibitors

Research Project

Project/Area Number 16390017
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionKumamoto University

Principal Investigator

YAMAGUCHI Yoshihiro  Kumamoto University, Graduate School of Medical and Pharmaceutical Sciences, Instructor, 大学院・医学薬学研究部, 助手 (10363524)

Co-Investigator(Kenkyū-buntansha) KUROSAKI Hiromasa  Kumamoto University, Graduate School of Medical and Pharmaceutical Sciences, Associate Professor, 大学院・医学薬学研究部, 助教授 (70234599)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 2005: ¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 2004: ¥8,500,000 (Direct Cost: ¥8,500,000)
Keywordshydrolase / β-lactam antibiotics / substrate-recognition mechanism / inhibitor / site-directed mutagenesis / x-ray crystallography / kinetics
Research Abstract

Metallo-β-lactamase (IMP-1) poses a potential threat for clinical environment because of wide spread substrate specificity and lack of the available inhibitors. IMP-1 requires two Zn(II) ions to hydrolyze β-lactam antibiotics.
To study the effects on Zn(II) binding to IMP-1, we examined the kinetics of dissociation of Zn(II) from wild type IMP-1. From the kinetic experiments, the dissociation of Zn(II) ion from IMP-1 appeared to be two steps. Apo IMP-1 was prepared by the addition of EDTA directly to wild type IMP-1 at 30 ℃. Up to the addition of Co(II) to apo IMP-1 in 1:1, the absorption bands due to d-d transitions appeared. Further addition of Co(II) to apo IMP-1 up to 2:1 resulted in the increases in absorption at 344 nm, due to LMCT from thiolate of Cys221 to Co(II), and d-d transitions. These result suggest that the binding of Co(II) to apo IMP-1 proceeds stepwise : a Co(II) ion initially binds to the Zn1 site and then the second Co(II) ion binds to the Zn2 site.
We prepared two inhibitors, pentafluorophenyl 3-mercaptopropionate (PFMP, 1) and 3-(3-mercaptopropionylsulfanyl)propionic acid pentafluorophenyl ester (MPAP, 2) for one of MBLs, IMP-1. From the gel-filtration experiment of the enzyme-inhibitor complex, these compounds inhibited IMP-1 irreversibly. Moreover, X-ray crystallography revealed that inhibitor 2 covalently binds to IMP-1 to form an amide bond between the amino group (N^ζ) of Lys224 and the inhibitor.

Report

(3 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • Research Products

    (5 results)

All 2006 2005

All Journal Article (5 results)

  • [Journal Article] β-ラクタム剤耐性菌が産生するメタロ-β-ラクタマーゼの三次元構造に立脚した非可逆的阻害剤の開発2006

    • Author(s)
      山口 佳宏
    • Journal Title

      The Japanese Journal of Antibiotics 59(1)

      Pages: 51-56

    • NAID

      10017296202

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Journal Article] Structure-based drug design of the irreversible inhibitors of metallo-β-lactamases2006

    • Author(s)
      Yoshihiro Yamaguchi
    • Journal Title

      The Japanese Journal of Antibiotics 59(1)

      Pages: 51-56

    • NAID

      10017296202

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Probing the role of Asp120(81) of metallo-β-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography2005

    • Author(s)
      Yoshihiro Yamaguchi
    • Journal Title

      Journal of Biological Chemistry 280(21)

      Pages: 20824-20832

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Journal Article] Irreversible inhibition of metallo-β-lactamase (IMP-1) by 3-(3-mercaptopropionylsulfanyl)propionic acid pentafluorophenyl ester2005

    • Author(s)
      Hiromasa Kurosaki
    • Journal Title

      Angewandte Chemie International Edition 44

      Pages: 3861-3864

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Journal Article] Probing the role of Asp120(81) of metallo-β-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography2005

    • Author(s)
      Yoshihiro Yamaguchi
    • Journal Title

      Journal of Biological Chemistry (印刷中)

    • Related Report
      2004 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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