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 Km and enzymatic catalysis rate k2, for FDG hydrolysis to FMG by beta-galactosidase were obtained as 18.0 microM and 1.9 mumol.(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 microM), 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 (greater than Km), 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Huang
Z J
ZJ
Molecular Probes, Inc., Eugene, Oregon 97402.
D001667:Binding, Competitive; D002384:Catalysis; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes; D005470:Fluorometry; D005697:Galactosides / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005697:Galactosides; C038848:fluorescein-digalactoside; D001616:beta-Galactosidase