No intermediate channelling in stepwise hydrolysis of fluorescein di‐β‐D‐galactoside by β‐galactosidase

摘要: For the hydrolysis of two glycosidic bonds fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase from Escherichia coli, small [Hofmann, J. & Sernetz, M. (1983) Anal. Biochem. 131, 180-186] to dramatic [Huang, Z. (1991) Biochemistry 30, 8535-8540] deviations simple stepwise substrate-intermediate-product kinetics have been reported. Intermediate channelling, a preferred intermediate mono-beta-D-galactoside (FMG) formed FDG at active site and thus in favourable position for further reaction, has postulated. As there were reasons doubt previous findings conclusions, experiments repeated initial concentrations 7-200 microM, following FDG, FMG with reliable method, quantitative HPLC, completion reaction. The transient appearance substantial amounts also 200 microM already rules out existence most efficient channelling deduced Huang measurements initially developing fluorescence, incorrectly ascribed fluorescein. Redetermination Michaelis constants led much higher values than those reported previously. Fitting progress curves means nonlinear regression combined numerical integration rate equations resulted good fits normal mechanism, without any necessity assuming more complex course So one rare examples single enzyme-substrate encounter invalidated.