KINETIC FLUORESCENCE MEASUREMENT OF FLUORESCEIN DI-BETA-D-GALACTOSIDE HYDROLYSIS BY BETA-GALACTOSIDASE - INTERMEDIATE CHANNELING IN STEPWISE CATALYSIS BY A FREE SINGLE ENZYME

Kinetic fluorescence measurements were employed to quantitative to stepwise hydrolysis of fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase and the intermediate fluorescein mono-beta-D-galactoside (FMG) channeling. The kinetic parameters, Michaelis-Menten constant K(m) and enzymatic catalysis rate k2, for FDG hydrolysis to FMG by beta-galactosidase were obtained as 18.0-mu-M and 1.9-mu-mol.(min.mg)-1, respectively. The FMG intermediate is hydrolyzed via two modes: (1) FMG that is in free solution binding to the enzyme substrate binding site in competition with FDG and then being hydrolyzed (binding mode); (2) FMG being directly hydrolyzed into the final products of fluorescein and galactose before the FMG can diffuse away from the enzyme active site (channeling mode). The extent of the FMG channeling mode was found to depend on the FDG hydrolysis rate but to be independent of the free enzyme concentration. A channeling factor, defined as the ratio of the real FMG hydrolysis rate with both binding and channeling modes over that which would be observed with an exclusive binding mode, was used to quantitate the effect of the intermediate channeling. The FMG channeling factor was determined to be close to 1 at low FDG concentration (about 5.1-mu-M), where the slow FDG hydrolysis rate gives an ineffective channeling and where the FMG is then hydrolyzed mainly via the binding mode. However, the channeling factor dramatically increases at higher FDG concentrations (> K(m)), strongly indicating that the effective FMG channeling mode, resulting from the considerable FDG hydrolysis rate at high FDG concentrations, becomes a primary pathway to channel a steady system hydrolysis with a high rate. Under these conditions, FMG hydrolysis via the binding mode becomes insignificant compared to hydrolysis via the channeling mode as the substrate binding site of the enzyme is mostly occupied by FDG with high concentrations. The calculated FMG concentration formed as an intermediate in free solution in the stepwise hydrolysis is fairly low as a result of the effective FMG channeling at high FDG concentrations or, in the opposite case, relatively high in the case of weak FMG channeling at low FDG concentrations.