Author:
Carne T. J.,McKay D. J.,Flynn T. G.
Abstract
Phosphoglyceromutase (PGM) from chicken breast muscle was titrated with p-mercuribenzoate (PMB), 5,5′-dithiobisnitrobenzoate (Nbs2), N-ethylmaleimide (NEM), iodoacetate and iodoacetamide. The effect of all of the sulfhydryl reagents, with the exception of NEM was to cause a loss in enzymatic activity. Addition of KCN following reaction with Nbs2 resulted in the recovery of a small amount of enzymatic activity. In the absence of substrate (3-phosphoglyceric acid) or cofactor (2,3-diphosphoglyceric acid) and in the presence or absence of 6 M guanidine hydrochloride, six sulfhydryl groups per mole of enzyme were titrated with PMB. The total number of sulfhydryl groups determined by amino acid analysis of the performic acid oxidized and carboxymethylated enzyme was also found to be six. Disc gel electrophoresis in sodium dodecyl sulfate indicated that the enzyme is composed of subunits having the same molecular weights which suggests there are three sulfhydryl groups per subunit. Reaction with Nbs2 iodoacetate or iodoacetamide resulted in the modification of only two sulfhydryl groups. In each of the modifications, except that due to iodoacetamide both substrate and cofactor reduced the rate, but not the extent, of the reaction. Substrate was more effective than cofactor in reducing the rate of reaction with sulfhydryl reagents suggesting that a cysteine residue is involved in the binding of substrate. That the involvement is indirect is shown by the lack of protection offered by substrate and cofactor to reaction with iodoacetamide. Inactivation of PGM following titration with sulfhydryl reagents is not likely to be due to a conformational change since none of any significance was indicated by circular dichroism of the treated enzyme. This result was substantiated by the lack of a marked change in the fluorescence emission spectrum of the PMB treated enzyme.
Publisher
Canadian Science Publishing
Cited by
16 articles.
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