Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

Abstract

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate 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 in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which K d is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very 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 pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (K d ‫؍‬ 0.6 to 2.2 M, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism 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 the low affinity binding sites (B).Fumarase (fumarate hydratase, EC 4.2.1.2) catalyzes the reversible, stereospecific addition of water to fumarate to form L-malate (1). Being an enzyme of the citric acid cycle, it serves both catabolic and anabolic purposes and, as such, it is found within all living organisms. In eukaryotes, both mitochondrial fumarase (which is involved in the citric acid cycle) and the cytoplasmic enzyme are encoded by the same gene (2-4). These fumarase molecules are tetramers consisting of identical subunits of 50 kDa each, and their activity does not depend upon the presence of metal cations (1). In prokaryotes on the other hand, there are two distinct classes of fumarase molecules: class I fumarases are heat-labile and Fe 2ϩ -dependent, dimeric enzymes with subunits of 60 kDa each, having no obvious sequence homology to the eukaryotic enzymes, whereas class II fumarases are heat-stable, Fe 2ϩ -independent tetrameric enzymes with subunits of 50 kDa and showing extensive homology to the eukaryotic fumarases. Recently the three-dimensional structure of Escherichia coli class II fumarase has been unraveled (5-7). According to crystallograph...

Novel Role of Fumarate Metabolism in Dahl-Salt Sensitive Hypertension

“…Fumarase activity is conventionally measured in the direction of L-malic acid to fumaric acid due to the ease of the assay 16,17. Tissue specimens were homogenized and sonicated extensively in a cold Hepes solution (20 mM, pH 7.5, V/W 3:1).…”

Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

“…Pyromellitic acid (benzene-1,2,4,5-tetracarboxylic acid) has been previously reported as a weak inhibitor of some fumarate hydratase homologs (26), but it shows no inhibitory activity with respect to the M. tuberculosis enzyme. We have identified the first, to our knowledge, effective and selective small molecule inhibitor of the M. tuberculosis fumarate hydratase.…”

Stability, Subunit Interactions and Carbohydrate-Binding of the Seed Lectin from Pterocarpus angolensis

“…thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). This sample was supplemented with pig heart fumarase (200 kDa) that was purified and assayed as described [44].…”