The crystal structure of the tetrameric enzyme, fumarase C from Escherichia coli, has been determined to a resolution of 2.0 A. A tungstate derivative used in the X-ray analysis is a competitive inhibitor and places the active site of fumarase in a region which includes atoms from three of the four subunits. The polypeptide conformation is similar to that of delta-crystallin and is comprised of three domains. The central domain, D2, is a unique five-helix bundle. The association of the D2 domains results in a tetramer which has a core of 20 alpha-helices. The other two domains, D1 and D3, cap the helical bundle on opposite ends giving both the single subunit and the tetramer a dumbbell-like appearance. Fumarase C has sequence homology to the eukaryotic fumarases, aspartase, arginosuccinate lyase, adenylosuccinate lyase and delta-crystallin.

Fumarase C catalyzes the stereospecific interconversion of fumarate to L-malate as part of the metabolic citric acid or Kreb's cycle. The recent three-dimensional structure of fumarase C from Escherichia coli has identified a binding site for anions which is generated by side chains from three of the four subunits within the tetramer (Weaver et al., 1995). These same side chains are found in the three most highly conserved regions within the class II fumarase superfamily. The site was initially characterized by crystallographic studies through the binding of a heavy atom derivative, tungstate. A number of additional crystallographic structures using fumarase crystals with bound inhibitors and poor substrates have now been studied. The new structures have both confirmed the originally proposed active site, site A, and led to the discovery of a novel second binding site that is structurally nearby, site B. Site A utilizes a combination of residues, including H188, T187, K324, N326, T100, N141, S139, and S140, to form direct hydrogen bonds to each of the inhibitors. The A-site has been demonstrated by studying crystalline fumarase with the bound competitive inhibitors-citrate and 1,2,4,5-benzenetetracarboxylic acid. The crystal structure of fumarase C with beta-(trimethylsilyl)maleate, a cis substrate for fumarase, has led to the discovery of the second site or B-site. Sites A and B have different properties in terms of their three-dimensional structures. Site B, for example, is formed by atoms from only one of the subunits within the tetramer and mainly by atoms from a pi-helix between residues H129 through N135. The crystal structures show that the two locations are separated by approximately 12 A. A highly coordinated buried water molecule is also found at the active or A-site. The high-resolution crystal structures describe both sites, and atoms near the A-site are used to propose a likely enzyme/substrate complex.

The first structure of a new class of ATP-binding enzyme, PurM, has been solved and a model for the active site has been proposed. The structure is unprecedented, with an extensive and unusual sheet-mediated intersubunit interaction defining the active-site grooves. Sequence searches suggest that two successive enzymes in the purine biosynthetic pathway, proposed to use similar chemistries, will have similar ATP-binding domains.

Abstract:A search for the occurrence of the rare p-helix was performed with Iditis from the Oxford Molecular Group upon the Protein Data Bank. In 8 of the 10 confirmed crystal structures that harbor the p-helix, its unique conformation has been linked directly to the formation or stabilization of a specific binding site within the protein. In the discussion to follow, the role for each of these eight p-helices will be addressed in regard to protein function. It is clear upon closer examination that the conformation of the p-helix has evolved to provide unique structural features within a variety of proteins.