| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
In order to clarify the significance of conformational fluctuation of an enzyme in the catalytic mechanism, the dependence of rate constants for hydrolyses catalyzed by various serine proteases on medium viscosity were investigated. Acylation of α-chymotrypsin by a p-nitroanilides is hardly affected by medium viscosity, while that by a p-nitrophenyl ester decreases with increase in medium viscosity. This tendency is also observed in β-trypsin-catalyzed hydrolyses. For these enzymes, the conformational effect of the enzyme exists in formation of a tetrahedral intermedite, whereas its breakdown is almost free from the conformational effect. On the other hand, in addition to the acylation of subtilisin Carlsberg by p-nitrophenyl esters, that by p-nitroanilides is also influenced by medium viscosity. This suggests that both formation of (ES)_T and following breakdown of it are assisted by the conformational change of the enzyme. These results reveal that, although serine proteases have the same catalytic mechanism each other, there have some differences in the conformational effects during the catalytic processes.
In order to extend this method to the enzymes other than proteases, several novel reductases were isolated and purified from the cells of baker's yeast, which catalyze the asymmetric reduction of a carbon-carbon double bond in nitroalkenes and α,β-unsaturated ketones.
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