Kinetic analysis of urea-inactivation of beta-galactosidase in the presence of galactose |
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Authors: | Chilaka Ferdinand C Nwamba Charles O |
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Institution: | Department of Biochemistry, University of Nigeria, Nsukka, Nigeria. fc_chilaka@ibb.ut.ac.ir |
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Abstract: | 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. |
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