Z theor BioL (1989) 137, 171-189 Space-filling Effects of Inert Solutes as Probes for the Detection and Study of Substrate-mediated Conformational Changes by Enzyme Kinetics: Theoretical Considerations DOUGLAS A. BERGMAN AND DONALD J. WINZORt Department of Biochemistry, University of Queensland, St. Lucia, Queensland 4067, Australia (Received 8 August 1988) From expressions derived for the space-filling effects of small inert solutes on kinetic parameters for univalent enzymes undergoing isomerizations that are substrate- induced and pre-existing, it is concluded that experimental observation of an enhanced maximal velocity in the presence of inert solute can only reflect the existence of the former type of conformational change; and that the isomerization must be governed by a relatively small equilibrium constant. Similar conclusions apply to multivalent enzymes exhibiting Michaelis-Menten kinetics. Extension of the theory, to provide quantitative expressions for multivalent enzymes has made possible the numerical simulation of thermodynamic non-ideality effects on systems conforming with the Monod and Koshland models of allostery. In that regard the simulated Scatchard plots for the two models differ sufficiently in form to suggest that detailed examination of the space-filling effects of small solutes on the kinetics of an allosteric enzyme may, under favourable circumstances, allow identification of the appropriate allosteric mechanism. Finally, these considerations of thermody- namic non-ideality in relation to the kinetics of allosteric enzymes have revealed formal similarities between the consequences of space-filling by inert solutes and the specific effects of allosteric activators or inhibitors. Attention is drawn to the possible implications of this observation in relation to the functioning of allosteric enzymes in vivo, where catalytic performance may be modified by factors no more specific than the ability of unrelated solutes to occupy space in the highly concen- trated cellular environment. 1. Introduction Despite the fact that protein isomerization, be it pre-existing (Monod et al., 1965) or ligand-induced (Koshland et al., 1966), is frequently invoked as the molecular interaction responsible for many allosteric effects, there is very little direct evidence for the equilibrium co-existence of different conformational states of an enzyme or protein. Furthermore, in the few instances where substrate- or effector-mediated changes in protein conformation have been observed (e.g. Perutz et al., 1964; Gerhact & Schachman, 1968; Oberfelder et al., 1984a, b), assignment of the isomerization equilibria as ligand-induced or pre-existing has remained uncertain. This indecision t To whom all correspondence should be addressed at Department of Biochemistry, University of Queensland, St. Lucia, QId. 4067, Australia. 171 0022-5193/89/060171 + 19 $03.00/0 © 1989 Academic Press Limited