Fumarate hydratase

Fumarate hydratase, also called fumarase, is the enzyme that reversibly interconverts fumarate plus water with L-malate. The activity is EC4.2.1.2:In the hydration direction, water adds across the carbon-carbon double bond of fumarate. In the reverse direction, water is eliminated. Its lyase classification describes this transformation; the mere presence of water does not make the reaction hydrolysis.^[ref]

For an invented inventory, begin with ten micromoles of fumarate and let six micromoles undergo hydration. Four micromoles of fumarate remain and six micromoles of L-malate form. Each retains a four-carbon skeleton, so the counted carbon inventory remains $4(4+6)=40$ micromoles of carbon atoms. The chosen extent does not determine chemical equilibrium or enzyme kinetics.

A 1995 X-ray crystallography study of Escherichia coli fumarase C found a four-subunit assembly at $2.0\text{A˚}$ resolution. A competitive tungstate derivative helped locate the active site, which included atoms from three of the four protein subunits.^[ref] The example shows why counting subunits and locating a catalytic site are different tasks: several chains can cooperate to build one local reaction environment.

Fumarase C, often designated FumC, is a classII form of fumarate hydratase. It catalyzes reversible hydration of fumarate to L-malate. The protein designation should be tied to an organism: Escherichia coli fumarase C is a well-studied species-specific example, while the broader enzyme-activity name does not specify one protein sequence.^[ref]

The net substrate inventory makes the chemistry explicit:Four carbon atoms remain four; adding water changes hydrogen and oxygen content without changing the total charge. This hydration is chemically distinct from oxidation of L-malate to oxaloacetate. The enzyme accelerates a reversible reaction rather than making its net direction independent of conditions.^[ref]

The1995 X-ray crystallography study of the E.coli enzyme resolved an assembly of four identical protein subunits at2.0Å. Its proposed active site involved contributions from three of the four protein subunits; a local catalytic environment therefore need not lie inside just one polypeptide chain. A1996 follow-up with bound compounds distinguished the catalytic A-site from a nearby second binding site. These experiments also show why observing ligand binding does not by itself identify a catalytic site.^[ref][ref]

Escherichia coli fumarase C is a fumarate hydratase of Escherichia coli, commonly designated FumC. It catalyzes the reversible conversion between fumarate plus water and L-malate in the citric acid cycle. The name identifies a particular enzyme, while the broader activity name can cover related catalysts.^[ref]

The 1995 structure revealed a four-subunit assembly at $2.0\text{A˚}$ resolution, with the active site formed using atoms from three chains.^[ref] Further structures in 1996 used bound inhibitors and poor substrates to distinguish this catalytic A-site from a nearby B-site about $12\text{A˚}$ away. The second site was formed by one subunit rather than three.^[ref]

This gives a concrete interpretation problem for a binding experiment. Detecting a compound somewhere on the protein does not establish that it occupies the catalytic site. Comparing its position with substrate analogues, inhibitory behaviour and the multisubunit geometry supplies additional evidence. Similarly, a change in assembly could affect catalytic activity while retaining the same substrate-contacting amino acid residues. Structure and reaction-rate measurements describe complementary aspects of the working enzyme.