The role of the allosteric B site in the fumarase reaction.

Abstract

The role of a malate binding site in a concavity external to the more deeply situated active site has been a major mystery of the fumarase reaction. The malate, within 12 A of the active site, was bound by hydrogen bonds to two main-chain amides and to two basic residues, H129 and R126. Mutation of the His of this so-called B site of Escherichia coli fumarase had little effect on the overall initial rate kinetics of the enzyme, which has obscured an understanding of the critical role of the site. Contrary to the WT enzyme, which is rate-limited in the recycling of free enzyme isoforms that follows product release, the enzyme with both basic residues modified is rate-limited in the product release step itself. A loss of complexity in the mutated, but still functional, step is indicated by a greatly reduced sensitivity of its rate to changes in temperature. Unlike the inhibition by glycerol shown with normal enzyme and attributed to a viscogenic effect on the recycling rate, the product-release step of the B-site mutants is accelerated by glycerol, suggestive of a structural effect on the 12-A space between the A and B sites. It is proposed that the "extra" malate represents a stage in the transfer of substrate and product between the solvent and the "buried" active site of the enzyme.