Evidence for a single essential thiol in the yeast hexokinase molecule.
In yeast hexokinase B, two thiols per monomer appeared to be essential when enzymic inactivation was produced by the concurrent alkylation of both of them, by several reagents including the affinity reagent N-bromoacetyl-2-D-galactosamine. However, it is shown that only one of these thiols is actually essential. Three of the four thiols present can be blocked by alkylation in the presence of a substrate in appropriate conditions, without loss of enzymic activity. Subsequently, in the absence of substrate, the affinity reagent reacts at the one remaining thiol, with complete inactivation. The same behavior can be obtained by reaction with iodoacetamide or by the formation of the -SCN group. The affinity reagent inactivates hexokinase B faster than does the isomeric glycosidic compound (glycosides being nonsubstrates), although the latter has twice the reactivity of the former toward glutathione. The reactions with alkylating agents, with or without substrate present, are used to classify the four thiols in the monomer. The temperature dependence of the alkylation of the essential thiol provides evidence for a transition in the molecule at about 31 degrees C. The inactive monomer containing the -SCN group can regenerate, by thiolysis, active enzyme with the thiol free. It can also perform an intramolecular cleavage of the chain. The latter reaction was used to locate the essential cysteine residue in the chain, at 80% of the length from the N terminus.
D000116:Acetylgalactosamine / Q000031:analogs & derivatives; D000345:Affinity Labels; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D006593:Hexokinase; D007460:Iodoacetamide; D007461:Iodoacetates; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013816:Thermodynamics; D013861:Thiocyanates
D000345:Affinity Labels; D007461:Iodoacetates; D013861:Thiocyanates; D004228:Dithionitrobenzoic Acid; D006593:Hexokinase; D003545:Cysteine; D000116:Acetylgalactosamine; D007460:Iodoacetamide