PIG-HEART FUMARASE CONTAINS 2 DISTINCT SUBSTRATE-BINDING SITES DIFFERING IN AFFINITY

A eukaryotic fumarase is for the first time unequivocally shown to con tain two distinct substrate-binding sites. Pig heart fumarase is a tet rameric enzyme consisting of four identical subunits of 50 kDa each. B esides the true substrates L-malate and fumarate, the active sites (si tes A) also bind their analogs D-malate and oxaloacetate, as well as t he competitive inhibitor glycine, The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-mala te and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (K-d being i n the millimolar range) is 1-2 orders of magnitude lower than the affi nity of sites A (of which hh is in the micromolar range). However, sat urating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays v ery special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time, Four molecules of py romellitic acid were found to bind per molecule fumarase, and the affi nity of the enzyme for this ligand is very high (K-d = 0.6 to 2.2 mu M , depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosin e residue is found to be located in site A, whereas an essential methi onine residue resides in or near site B, Upon limited proteolysis, a p eptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small l ocal conformational changes in the enzyme are picked up by circular di chroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating th e low affinity binding sites (B).