The substrate specificity of fumarase.

The substrate specificity of fumarase has been studied by measuring the ability of the enzyme to hydrate or dehydrate derivatives of fumarate and L-malate. Fumarase was observed to hydrate fumarate and its derivatives in the order, fluorofumarate > fumarate > chlorofumarate > bromofumarate > acetylenedicarboxylate > iodofumarate > mesaconate. With the exception of fluorofumarate, water was added trans to these substrates to produce the three+?substituted derivatives of L-malate. Fluorofumarate was hydrated to give cu-fluoromalde, which spontaneously decomposed to oxalacetate. Acetylenedicarboxylate was also hydrated by fumarase to oxalacetate. The enzymecatalyzed dehydration of the L-malate analogues was shown for L-threo-chloromalate, (-)threo-bromomalate, L-tartrate, and threo-hydroxyaspartate.’ In addition to defining more precisely the substrate specificity of fumarase, these studies suggest that steric hindrance by the substituent groups to proper substrate binding is primarily responsible for the observed order of reactivity among the new substrates reported here. Furthermore, the monosubstituted derivatives of fumarate may form two nonequivalent complexes with the active site, depending upon the nature of the substituent group. Finally, the region of the active site catalyzing the addition of hydrogen may be sterically less specific than the region catalyzing the addition of the hydroxyl group.