Reversible inactivation and dissociation of yeast hexokinase.

Recent’ work on the reactivation and reconstitution of several denatured enzymes has provided increasing understanding of the process of reversible protein denaturation. Studies on ribonuclease by Anfinsen et al. (1) and White (2) have convincingly demonstrated that under favorable conditions a complet,ely denatured protein can resume the active three-dimensional configuration of the native molecule. Essentially similar results have been obtained in the case of lysozyme by Isemura et al. (3, 4) and Goldberger and Epstein (5) and on Taka-amylase by Isemura et ul. (3). These examples provide justification for the view that t,he secondary and tertiary structure of a protein molecule is a result solely of the intrinsic thermodynamic properties of its amino acid sequences (1, 2). These conclusions based on work with single chain proteins have recently been extended to complex, multichain proteins. Thus, reports by Stellwagen and Schachman (6), Deal, Rutter and Van Holde (7), and Hass and Lewis (8) on rabbit muscle aldolase and by Samejima and Yang (9) on catalase illustrate the ability of denatured and dissociated polgl)eptide chains of a protein molecule to reassemble and refold under appropriate conditions to give the biologically active conformation of the native molecule. Sedimentation and diffusion studies on yeast hexokinase by Kunitz and XlcI>onald (10) indicated that the enzyme has a molecular weight of 96,600 at pH 5.5. These workers, quoting the data obtained by -1. Rothen, were the first to report that hexokinase exhibits a second slowly moving peak in the ultracentrifuge at pH 6.0, which would now be interpreted to represent the half molecule of the enzyme. They, however, interpret,ed this as an “effect, of ultracentrifugation on the homogeneity of the hexokinase protein.” 11ore recently Schachman (11) has I)rovided further evidence for the dissociation of hexokinase into half-molecules without loss of activity, under influence of pH, phosphate ions, and glucose.’ Ramrl, Stellwagen, and Schachman (12) have also reported that treatment of hexokinase with a detergent caused dissociation into al)prosimately quarter-molecules, which were inactive. In this (Lommunication we present experiments to show that the inactivation of yeast hexokinase in acid and alkali is accompanied by its dissociation into inactive quarter molecules which reassociate at neutral pH to produce the active enzyme with physical, chemical, and biological properties close t.o those of