This page is for Alex Waters' fumarase page.
is a tetrameric enzyme which is responsible for catalyzing the hydration and dehydration of L-malate and fumarate. As an enzyme that has been well characterized in the lab, many crystallographic studies have been done to describe the active site of this enzyme. Several of these studies have been done using inhibitors like pyromellitic acid and beta-trimethylsilyl maleate. These studies produced different results. An area called the "tungsten site", which formed between atoms of three of the four subunits was determined to be the binding site of some of these inhibitors and was called the "A-site". This site exists deep in the enzyme. However, other studies provided data indicating another site, called the "B-site" which also bound the inhibitors and was composed of atoms from one subunit only and is closer to the surface of the enzyme. Both the A-site and the B-Site make use of a Histidine residue to catalyze the reaction. In order to determine the correct active site of fumarase, two mutants were created which altered the histidines responsible for catalyzing the reaction at each site. was expected to change catalytic activity if the A-site was the active site and would change catalytic activity if the B-site was the active site. By measuring the activity of each mutant and comparing it to the wild-type enzyme, is was determined that the A-site of fumarase is the active site, as the activity of this mutant was nearly 200 times less than the wild type, while the B-site's activity remained roughly equal to the wild-type. Crystal structures of these two mutants further confirmed that the A-site is the .
Function
Disease
Relevance
Structural highlights
The active site contains a water molecule which assists in catalyzing the reaction by abstracting a proton from the C3 of malate. Furthermore, the Histidine 188 in the A-site is responsible for binding the C4 site of malate. This atom is the site of the double-negative charge on the transition state and is stabilized through hydrogen bonds to Ser98, Thr100 and Asn141 and Coulombic interactions. The H188 atom donates a proton to the active site water molecule which stabilizes the double negative charge on the transition state with its new positive charge. Thus, H188 is involved in activating the water molecule to catalyze the reaction. shows the His188 mutant with L-malate bound to the putative active site. shows the H129 mutant. shows the full quaternary structure of fumarase with all His129 and His188 labeled.
