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The alpha-amidating enzyme. The intermediary reaction steps and the structure/function relationship as a bifunctional enzyme.

Research Project

Project/Area Number 05680558
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Functional biochemistry
Research InstitutionKurume University School of Medicine

Principal Investigator

NOGUCHI Masato  Kurume University School Medicine, Medical Biochemistry, Professor, 医学部, 教授 (10124611)

Co-Investigator(Kenkyū-buntansha) OHNAMI Tetsuo  Fukushima Medical College, Chemistry, Associate Professor, 医学部, 助教授 (70137008)
TSUKAMOTO Tetsuro  Fukushima Medical College, Neurology, Associate Professor, 医学部, 助教授 (20171978)
Project Period (FY) 1993 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsAmidating enzyme / Peptide amide / Isotope effect / Multiple sclerosis / 多発生硬化症 / C末端アミド化酵素 / 2頭酵素
Research Abstract

Peptidylglycine alpha-amidating activity catalyzes the oxidation of a C-terminally glycine-extended peptide to a desglycine alpha-amidated peptide at the expense of ascorbate and O_2 in the presence of copper. The reaction involves oxidative N-dealkylation within the terminal glycine, retaining the glycine N and releasing the remainder as glyoxylate. We revealed that the reaction consists of two steps via a carbinolamide as an intermediate (peptidyI alpha-hydroxyglycine), and also that two separate enzymes derived from a common precursor protein, peptidylglycine alpha-hydroxylase (PH) and peptidylamidoglycolate lyase (PGL), catalyze these steps, formation of the carbinolamide and its conversion into the alpha-amide and glyoxylate, respectively. The goals of the projects were (1) to clarify whether an alpha-carbon radical of glycine is involved in the formation of the carbinolamide intermediate by investigating isotope effects on the PH reaction, and (2) to construct an expression vecto … More r that is able to secrete the precursor protein into medium in order to probe the structure/function relationship as a bifunctional enzyme. Along with these projects, we have obtained the following results. (1) The hydroxylation of glycine is a ping-pong Bi Bi reaction in terms of ascorbate and the substrate (D-Tyr-Val-Gly). The deuterium effects on V/K and Vmax, using D-Tyr-Val-Gly-2,2-d2, are about 1.2, far less than 5.7 which has been reported by Kizar et al. Much more detailed kinetic study with tritium-labeled substrate is on going in order to learn the true isotope effect values, ^DV/K and ^DVmax. (2) The cDNA library has been prepared from rat atrium. The cDNA clone encoding the precursor protein is in search. (3) We have found the amidating activity in the cerebrospinal fluids (CSF) is increased in patients with multiple sclerosis, especially during the active stage, compared with that in patients with other neurological diseases. This suggests that the amidating activity in CSF could be an indication reflecting the physiological and morbid states of the central nervous system. Less

Report

(3 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] 野口正人: "ペプチドホルモンの生合成-C末端アミド化酵素の性質と反応機構" 久留米医学会雑誌. 56. 497-503 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 野口正人: "多発性硬化症の髄液アミド化酵素活性とその推移" 生化学. 66. 791- (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tetsuro Tsukamoto: "Paraneoplastic cereb-ellar degeneration and limbic encephalitis in a patient with adenocarcinoma of the colom." Journal of Neurology,Neurosurgery and Psychiatry. 56. 713-316 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Tetsuro Tsukamoto: "Peptidylglycine α-amidating monooxygenase(PAM)activity is increased in cerebrospinal fluid of patients with multiple sclerosis." Internal Medicine. (印刷中). (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] M.Noguchi: "Peptidylglycine alpha-amidating enzyme. Characterization and the reaction mechanism." Journal of Kurume Medical Association. 56. 497-503 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] M.Noguchi: "alpha-Amidating activities in cerebrospinal fluids of multiple sclerosis patients" Seikagaku. 66. 791 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] T.Tsukamoto: "Paraneoplastic cerebellar degeneration and limbic encephalitis in a patient with adenocarcinoma of the colon." Journal of Neurology, Neurosurgery and Psychiatry. 56. 713-716 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] T.Tsukamoto: "Peptidylglycine alpha-amidating monooxygenase (PAM) activity is increased in cerebrospinal fluid of patients with multiple sclerosis." Internal Medicine. (in press). (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 野口正人: "ペプチドホルモンの生合成-C末端アミド化酵素の性質と反応機構" 久留米医学会雑誌. 56. 497-503 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] 野口正人: "多発生硬化症の髄液アミド化酵素活性とその推移" 生化学. 66. 791 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Tetsuro Tsukamoto: "Paraneoplastic cerebellar degeneration and limbic encephalitis in a patient with adenocarcinoma of the colon." Journal of Neurology, Neurosurgery and Psychiatry. 56. 713-716 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] Tetsuro Tsukamoto: "Peptidylglycine α-amidating monooxygenase (PAM) activity is increased in cerebrospinal fluid of patients with multiple sclerosis" Internal Medicine. 印刷中. (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Masato Noguchi: "The source of oxygen atom in the alpha-hydroxyglycine intermediate of the peptidylglycine alpha-amidating reaction" Biochemical Journal. 283. 883-888 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] Hiroshi Okamoto: "Peptide alpha-amidation is achieved by distinct enzyme molecules in a single mRNA" The Proceedings of the Yamada Conference XXVII. 113-116 (1991)

    • Related Report
      1993 Annual Research Report
  • [Publications] Kenichi Takahashi: "Peptidylglycine alpha-amidating reaction:Evidence for a two-step mechanism involving a stable intermediate at neutral pH" Biochemical and Biophysical Research Communications. 169. 524-530 (1990)

    • Related Report
      1993 Annual Research Report
  • [Publications] Masahiro Tajima: "The reaction product of peptidylglycine alpha-amidating enzyme is a hydroxyl derivative at alpha-carbon of the carboxyl-terminal glycine" Journal of Biological Chemistry. 265. 9602-9605 (1990)

    • Related Report
      1993 Annual Research Report
  • [Publications] Masato Noguchi: "Rat peptidylglycine alpha-amidating enzyme:The relation between activities at neutral and alkaline pH values" Archives of Biochemistry and Biophysics. 275. 505-513 (1989)

    • Related Report
      1993 Annual Research Report
  • [Publications] 野口,正人: "ペプチドホルモンの生合成-C末端アミド化酵素の性質と反応機構-" 久留米医学会雑誌. 56. 497-503 (1993)

    • Related Report
      1993 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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