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ABSTRACT Although the idea that electrostatic potentials generated by enzymes can guide substrates to active sites is well established, it is not always appreciated that the same potentials can also promote the binding of molecules other than the intended substrate, with the result that such enzymes might be sensitive to the presence of competing molecules. To provide a novel means of studying such "electrostatic competition" effects, computer simulation methodology has been developed to allow the diffusion and association of many solute molecules around a single enzyme to be simulated. To demonstrate the power of the methodology, simulations have been conducted on an artificial fusion protein of citrate synthase (CS) and malate dehydrogenase (MDH) to assess the chances of oxaloacetate being channeled between the MDH and CS active sites. The simulations demonstrate that the probability of channeling is strongly dependent on the concentration of the initial substrate (malate) in the solution. In fact, the high concentrations of malate used in experiments appear high enough to abolish any channeling of oxaloacetate. The simulations provide a resolution of a serious discrepancy between previous simulations and experiments and raise important questions relating to the observability of electrostatically mediated substrate channeling in vitro and in vivo.
INTRODUCTION
A number of enzymes are known to exert electrostatic forces on substrates that promote their productive encounter with the active site (Blacklow et al., 1988; Getzoff et al., 1992; Radic et al., 1997). The relatively long-range nature of electrostatic interactions means that they can operate at distances beyond those at which the atomic details of the molecules are important. As a result, the same attractive forces that act on an intended substrate are equally capable of operating on "incorrect" molecules that happen to share the same essential charge features of the real substrate. As the concentration of these other molecules is increased, it becomes increasingly likely that the substrate will encounter "electrostatic competition" during the process of binding to the enzyme. If the concentration is high enough, electrostatically driven association might be completely suppressed, with the result that the substrate associates at a rate more consistent with a random collision with the enzyme. A familiar manifestation of such a phenomenon is the sensitivity of enzyme-substrate and protein-protein association kinetics to ionic strength....