The number of substrate- and inhibitor-binding sites of fumarase.

The reversible binding to fumarase of the competitive inhibitors, tram+aconitate and citrate, and of the natural substrates, fumarate and L-malate, was studied by the method of equilibrium dialysis. The binding of the enol tautomer of oxalacetate, a secondary substrate of fumarase, was measured spectrophotometrically. The results of these studies indicate that there are four substrateor inhibitor-binding sites per tetrameric molecule of enzyme, or an average of one site per polypeptide chain subunit. The Michaelis constants (K,) and inhibition constants (K,) for these compounds were found to be in close agreement with the dissociation constants calculated from the binding studies. This correspondence strongly suggests that the four binding sites measured are also catalytic sites.