Sandbox Wabash 03 fumarase

Fumarase is a tetrameric enzyme that catalyzes the dehydration of L-malate to Fumarate and vice versa. It does so by deprotonating the C3 carbon of L-malate and removing an OH group from the C2 carbon, or by adding a water across the double bond of fumarate.

Fumarase has two sites where substrates can bind. The first site, which is referred to as "site A" in a Weaver et al. 1997 study ^[1] is deeper in the enzyme structure than the second site, "site B". In addition it is composed of atoms from 3 of the four subunits, where as site B is completely composed of atoms from a single sub unit. ^[2]

A dilemma arose over which was the active site. A 1996 crystallographic study showed L-malate and beta-trimethylsilyl maleate in site B.^[3] However, it was suspected that site A was the active site, because no active monomeric unit of fumerase had ever been described. Furthermore, in the previously mentioned study, citrate, which is a known competitive inhibitor of fumarase activity, was used at high concentration as the crystallizing agent. Hence, this would explain L-malate's association at site B rather than A. In a 1997 study by Weaver et. al, mutations were introduced at each site. Since Histidine was suggested to be one of the bases participating in the catalytic activity of fumarase Weaver et. al made site A fumarase mutant H188N and site B fumarase mutant H129N examine which of the sites might be the active site. In short, their study showed that the change of Histidine 188 to an asparagine showed a large decrease in specific activity for fumarase, while the same change at Histidine 129 showed essentially no effect. ^[4] This showed that site A was the active site for fumarase.

The active site contains a number of different, charged residues, including S98, T100, N141, H188, K324, N326, and E331. In addition, four of these are hydrogen bonded to a water in the pocket of the active site. These are S98, T100, N141, and H188. ^[5] The many charged, and hydrogen bond capable residues in the active site suggest a mechanism in which they are involved in stabilizing the charged, substrate intermediate. Furthermore the coordination of water in the active site is likely important since the addition of water is involved in the reverse reaction.

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You may include any references to papers as in: the use of JSmol in Proteopedia ^[6] or to the article describing Jmol ^[7] to the rescue.

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