Kinetic analysis of urea-inactivation of β-galactosidase in the presence of galactose

Abstract

The effect of galactose on the inactivation of purified β-galactosidase from the black bean, Kestingiella geocarpa, in 5 M urea at 50°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 β-D-galactopyranoside (PNPG) as substrate, S. The inactivation data were analysed using the Tsou equation and plots. Plots of ln([P]_∞ – [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]_∞ vs [S] were used to determine the mode of inhibition of the enzyme by galactose, and slopes and intercepts of the 1/[P]_∞ 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.

Keywords::

• β-galactosidase
• urea
• inactivation
• denaturation
• galactose
• inhibition
• folding intermediates