The Thiol Groups of Fumarase

Abstract

Several reagents have been employed to determine the role of the thiol groups in fumarase. The nature of the reaction of these reagents indicates that the thiol groups are not associated with active site structures but are buried in a hydrophobic environment in the interior of the enzyme. Several observations are in accord with this view. The rate of reaction of the thiol groups is much slower in the native, active enzyme than in the unfolded, inactive molecule. The rate of reaction is also a function of the polarity of the thiol reagent. Uncharged aliphatic reagents react faster than more polar reagents. The rate of reaction of a series of alkyl mercury nitrates increases in proportion to the number of methylene groups in the alkyl side chain. In dilute aqueous solutions of aliphatic alcohols, the rate of reaction of p-chloromercuribenzoate with the thiol groups increases with alcohol concentration and the number of methylene groups in the alcohol. The rate of reaction of iodoacetate at pH 6.5 is minimal at 23.5° but increases at lower or higher temperatures. An increase in reactivity at low temperatures appears to occur as a consequence of a decreased strength of hydrophobic interactions at low temperatures.