1988 Fiscal Year Final Research Report Summary

Analysis of The Mechanism of Nitrgenase by using Monoclonal Antibodies

Research Project

Project/Area Number	62560074
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	応用生物化学・栄養化学
Research Institution	The University of Tokyo
Principal Investigator	ONODERA Kazukiyo The University of Tokyo Department of Agricultural Chemistry, 農学部, 助教授 (90012773)
Project Period (FY)	1987 – 1988
Keywords	protein engineering / nitrogen fixation / monoclonal antibody / DNA sequencing / metal enzyme / 多基質酵素
Research Abstract	Nitrogenase is the N_2 -fixing enzyme system composed of two proteins Componentl and Componetll. For enzymatic activity electrons are funneled through the componentll to the Componentl where substrate reduction occurs. The Componentl,a Mo-Fe protein,has a wide range of the substrate specificity. It reducts N_2,CN and C_2H_2 , and it also reducts H^+ to H_2 when no other substrate exists. We cloned the DNA fragment coding for the region of Componentl , which contained point mutations , from a mutant of A,vinelandii requiring W instead of mo which we previously obtained. The sequence is under analysis. We expect that the positions of mutations in primary structure of Componentl will be determined which cause the change of metal requirment from Mo to W. We have already obtained anti-Componentl monoclonal antibodies,and it was shown that one of the antibodies selectively inhibited only C_2H_2 reduction,and another did only H^+ reduction. This observation implied that the specificity of binding sites of these monoclonal antibodies caused the specific patterns of inhibition. Then we expected that determination of antigenic determinantal regions of them on the Componentl would give some ackowledgement about active centers for various enzymatic activities. And we conformed by using expression vectors which contained the DNA fragment coding for Componentl that antigenic determinantal regions of the antibodies, which had different patterns of inhibition,differed.