|Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥1,200,000 (Direct Cost: ¥1,200,000)
In this research project, we proposed a hypothesis on the molecular mechanism of the enzyme-catalyzed acceleration, designated by the equation, E(] SY.dblharw.[)E* * E', where E is native state, E' is denaturated state, and E* is an intermediate. Rate equation of denaturation is represented as follows, v = k' [E*], where k' is rate constant. E* is supposed to be active, thus a problem is how to make clear it on the experiments. On the basis of the acceleration model, a substrate(S) is the decisive key to solve the problem ; rate of denaturation (v) is very dependent on the concentration of substrate [S] , when E* is active. However, the rate is independent on [S] , when E is active. In the experiments, a transition state analogue glucanolacton was used for S, because a substrate is catalyzed with the enzyme during observation of denaturation.
Denaturation of an enzyme glucoamylase from Rhizopus niveus, caused at 60ﾟC and pH 4.5, was observed by spectrophotometric procedures at 280nm. Rate of denaturation v was found to be dependent on [S] and a plot of DELTA v - [S] gives the dissociation constant of the gluconolatone-glucoamylase complex, Kd to be 3.8mM, which is nearly equal to the reported values (1.5 * 0.5mM). These findings suggest that E* is active form of the enzyme and E is a resting state for the enzyme-catalyzed activity.
We take notice on the near infrared(NIR), which is a useful strategy for specific transition of enzyme conformation, thus an apparatus of the NIR radiation was constructed using a halogen lamp unit and a spectromate, made of Shimadzu. Radiation effect on the acceleration is tested for alpha-amylase and tyrosinase.