2000 Fiscal Year Final Research Report Summary
Rational Design of a Catalytic Site in Myoglobin
| Project/Area Number |
10680575
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | Yamagata University (1999-2000)
Okazaki National Research Institutes (1998) |
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Principal Investigator |
OZAKI Shin-ichi Yamagata University, Associate Professor, 教育学部, 助教授 (40280581)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Yoshihito Institute for Molecular Science Professor, 分子科学研究所, 教授 (10201245)
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| Project Period (FY) |
1998 – 2000
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| Keywords | heme / peroxidase / myoglobin / heme oxygenase / oxidation |
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| Research Abstract |
In order to pursue structure-function relationships on the activation of peroxides, we have chosen to use sperm whale myoglobin as the framework for our molecular engineering studies. Comparison of crystal structures of myoglobin and the other enzymes like heme oxygenase or peroxidase reveals differences in arrangement of amino acid residues in heme active sites. On the basis of structural differences and the reaction mechanisms, we have converted myoglobin into an enzyme by alternation of the heme distal pocket via site-directed mutagenesis. Our achievements are summarized as follows.
1) The polarity as well as hydrogen bonding between oxygen molecule bound to the heme iron and His or Trp appears to be important in controlling the regiospecific heme degradation in the coupled oxidation of sperm whale myoglobin mutants.
2) The H93G myoglobin mutant was constructed to leave an open cavity capable of accommodating a variety of substituted imidazole (Im-X) as proximal ligands for the heme i
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ron. With increasing the electron donation by Im-X, the reaction of H93G(Im-X) Mb with hydrogen peroxide is facilitated ; however, the electron push effect is not as large as expected.
3) The H64D myoglobin mutant constructed to mimic the active site of chloroperoxidase from Caldariomyces fumago exhibits more than 300-fold better oxidation activity than the wild type. The results suggest that the polar active site increases the reactivity of the ferric myoglobin with hydrogen peroxide.
4) The F43W/H64L myoglobin mutant has been constructed to investigate effects of an electron rich oxidizable amino acid residue in the heme vicinity on oxidation activities of Mb. During the reaction of the mutant with m-chloroperbenzoic acid (mCPBA), Trp-43 is chemically oxidized presumably to quinone based on the results of MS and NMR analysis. The result could be a good model study for the biosynthesis tryptophan tryptophylquinone (TTQ), which is known as a covalently bound cofactor for methylamine dehydrogenase (MADH). Less
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Research Products
(16 results)
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[Publications] Murakami, T., Morishima, I., Matsui, T., Ozaki, S., Hara, I., Yang, H-J., Watanabe, Y.: "Effects of the Arrangement of a Distal Histidine on Regioselectivity of the Coupled Oxidation of Sperm Whale Myoglobin Mutants"Journal of the American Chemical Society. 121. 2007-2011 (1999)
Description
「研究成果報告書概要(欧文)」より
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