Inactivation of purified β-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 relevant ？？ ？？ and ？ m a x values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln ( [ ？？ ] ∞ ？ [ ？？ ] ？？ ) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1 / [ ？？ ] ∞ versus 1 / [ ？？ ] yielded ？？ + 0 and ？？ ′ + 0 , the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of ？？ + 0 and ？？ ′ + 0 versus [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. 1. Introduction A folded protein does not exist in a single conformation, rather as a set of related conformations whose interconversion involves the making and breaking of the weak (noncovalent) interactions such as hydrogen bonds, van der Waals, salt bridges, and hydrophobic interactions that stabilize the folded structure of the protein. The range and speed of interconversions between the conformations will depend on the magnitudes of the relevant energy barriers. In essence therefore, the observed three-dimensional structure of a protein should be viewed as a weighted average of all the conformations accessible on the time scale in question [1] in specific environments. The folding energy landscape theory or the folding funnel concept, used to explain the principle of folding, suggests that the most realistic concept of a protein is a minimally frustrated heteropolymer with a funnel-like rugged energy landscape biased towards the native structure [2–6]. The ruggedness of the energy landscape is biologically essential, controlling the distribution of protein conformations along the biologically relevant landscape, not necessarily around the funnel bottom [7]. The structures
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