Pressure effects on catalysis by thermolysin immobilized on polymer latices

Enzymes have been successfully immobilized onto various kinds of insoluble materials.Comparison of the characteristics of immobilized enzymes with those of free enzymes is very important to study both molecular and catalytic properties of enzymes.To carry out this comparison, the immobilization of enzymes onto polymer latices is advantageous because of the easiness of the direct spectrophotometric observation of enzymatic reaction process in the heterogeneous system.In a previous report, which was the first report examining pressure effects on the catalytic properties of immobilized enzymes, we showed the probability of the restriction of a conformational change of a physically "soft" enzyme by the immobilization onto polymer latices, which results in a decrease in the absolute value of the volume of activation (AV'), whereas the value of AVt of the "rigid" enzyme was not largely affected by the imm~bilization.~We also showed the small but definite interaction between an enzyme and an enzyme inhibitor which were bound onto different latex particle^,^ which was a model of the "cell-cell recognition" system.In this paper, we would like to report the kinetics of catalysis by the immobilized metal protease, thermolysin.Thermolysin was reported to have extremely large negative values of AV'.' As mentioned above, the reason of large negative AV' values have the probability to contain the factor of the conformational change during the enzyme reaction.6Because by the conformational change, polar parts of enzyme molecule are exposed to water during the catalytic process, which causes a stronger hydration and hence the large negative AV* value.To clarify this contribution to AV' values, we evaluated here AVt of thermolysin immobilized onto polymer latices. EXPERIMENTAL MaterialsThennolysin from Bacillus thennoproteolyticus rokko (EC 3.4.24.4) was purchased from Daiwa Kasei Co. (Osaka, Japan) and used without further purification.Bovine serum albumin (BSA, fat free, A-7511) was purchased from Sigma * To whom all correspondence should be addressed.