Kinetic analysis of urea-inactivation of beta-galactosidase in the presence of galactose.
The effect of galactose on the inactivation of purified beta-galactosidase from the black bean, Kestingiella geocarpa, in 5 M urea at 50 degrees C and at pH 4.5, was determined. Lineweaver-Burk plots of initial velocity data in the presence and absence of urea and galactose were used to determine the relevant K(m) and V(max) values, with p-nitrophenyl beta-D-galactopyranoside (PNPG) as substrate, S. The inactivation data were analysed using the Tsou equation and plots. Plots of ln([P](infinity) - [P](t) ) against time in the presence of urea yielded the inactivation rate constant, A. Plots of A vs [S] at different galactose concentrations were zero order showing that A was independent of [S]. Plots of [P](infinity) vs [S] were used to determine the mode of inhibition of the enzyme by galactose, and slopes and intercepts of the 1/[P](infinity) vs. 1/[S] yielded k(+0) and k '(+0), the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k(+0) and k '(+0) vs. galactose concentrations showed that galactose protected the free enzyme and not the enzyme-substrate complex against urea inactivation via a noncompetitive mechanism at low galactose concentrations and a competitive pattern of inhibition at high galactose concentrations. The implication of the different modes of inhibition in protecting the free enzyme was discussed.
Chilaka
Ferdinand C
FC
Department of Biochemistry, University of Nigeria, Nsukka, Nigeria. fc_chilaka@ibb.ut.ac.ir
Nwamba
Charles O
CO
D004791:Enzyme Inhibitors / Q000494:pharmacology; D005690:Galactose / Q000378:metabolism* / Q000494:pharmacology; D018525:Germination; D007700:Kinetics; D008954:Models, Biological; D012639:Seeds / Q000201:enzymology; D014508:Urea / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D014508:Urea; D001616:beta-Galactosidase; D005690:Galactose