SOLVENT AND VISCOSITY EFFECTS ON THE RATE-LIMITING PRODUCT RELEASE STEP OF GLUCOAMYLASE DURING MALTOSE HYDROLYSIS

Release of product from the active site is the rate-limiting step in a number of enzymatic reactions, including maltose hydrolysis by glucoa mylase(GA). With GA, an enzymatic conformational change has been assoc iated with the product release step. Solvent characteristics such as v iscosity can strongly influence protein conformational changes. Here w e show that the rate-limiting step of GA has a rather complex dependen ce on solvent characteristics. Seven different cosolvents were added t o the GA/maltose reaction solution. Five of the cosolvents, all having an ethylene glycol base, resulted in an increase in activity at low c oncentration of cosolvent and variable decreases in activity at higher concentrations. The increase in enzyme activity was dependent on poly mer length of the cosolvent; the longer the polymer, the lower the con centration needed. The maximum increase in catalytic activity at 45 de grees C (40-45%) was obtained with the three longest polymers (degree of polymerization from 200 to 8000). A further increase in activity to 60-65% was obtained at 60 degrees C. The linear relationship between In(k(cat)) and (viscosity)(2) obtained with all the cosolvents provide s further evidence that product release is the rate-limiting step in t he GA catalytic mechanism. A substantial increase in the turnover rate of GA by addition of relatively small amounts of a cosolvent has pote ntial applications for the food industry where high-fructose corn syru p (HFCS) is one of the primary products produced with GA. Since maltod extrin hydrolysis by GA is by far the slowest step in the production o f HFCS, increasing the catalytic rate of GA can substantially reduce t he process time.