Kinetic Analysis of Guanidine Hydrochloride Inactivation ofβ-Galactosidase in the Presence of Galactose

Abstract

Inactivation of purifiedβ-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevantKmandVmaxvalues, with p-nitrophenylβ-D-galactopyranoside (pNPG) as substrate,S. Plots of ln([P]∞−[P]t)against time in the presence of GdnHCl yielded the inactivation rate constant,A. Plots ofAversus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing thatAwas dependent on [S]. Slopes and intercepts of the1/[P]∞versus1/[S]yieldedk+0andk'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots ofk+0andk'+0versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed.