Kinetic Studies of the Reactivity of the Sulfhydryl Groups of Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract

The reaction of sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase from rabbit and pig muscles with a large molar excess of 5,5'-dithiobis(2-nitrobenzoate) (Nbs,) shows threephasic pseudo-first-order kinetics. Since the fastest reaction between active cysteine-149 and Nbs, is apparently biphasic, half-of-the-sites reactivity towards Nbs, is suggested. Further sulfhydryl groups become reactive as an effect of conformational changes in the protein molecule after formation of a mixed disulfide on cysteine-149. In the presence of 40 mM borate the reaction is biphasic only, and two sulfliydryl groups per subunit react very quickly.The bound NAD' is only partially released even after a long reaction with Nbs,. It was demonstrated that the two NAD+ binding sites with the highest dissociatio; constants have no significant effect on the reaction between cysteine-149 and Nbs,.Glyceraldehyde-3-phosphate dehydrogenase {D-glyceraldehyde-3-phosphate : NAD' oxidoreductase (phosphorylating)] is a glycolytic enzyme playing a key role in both glycolysis and gluconeogenesis. It is composed of four identical subunits of molecular weight 36000 [l]; the amino acid sequence of the enzymes from pig and lobster muscles [2] from yeast [3] and partially from human muscle [4] is known. The enzymes from rabbit and pig muscles are almost identical in sequence [2] and crystallise with 3 -3.5 mol of NAD' whose binding shows negative cooperativity [5,6]. Both enzymes have 16 cysteines in one tetramer, four of which are especially reactive. They are in position 149 and are essential for the enzymic activity, forming an acyl complex with the substrate. The remaining sulfhydryl groups have different reactivity, depending on the reagent used and on other factors including the structural state of the protein molecule and the conditions (buffer, ionic strength). There are some suggestions that cysteine-153, which can form a disulfide bridge with cysteine-149 [7], reacts with p-hydroxymercuribenzoate as the second one [8].Different aspects of the reaction with 5,5'-dithiobis-(2-nitrobenzoate) (Nbs,), a reagent used for the determination of sulfhydryl groups in proteins glyceraldehyde-3-phosphate dehydrogenase, 8 mol of 2-nitro-5-mercapto-benzoate (Nbs) are formed and half of the activity is restored during incubation owing to S -S bridge formation in the other half of enzyme molecules. Harrigan and Trentharn [12] assumed that NAD' dissociates from the lobsteienzyme before the reaction with Nbs,. In this case the rate of NAD' dissociation is rate-limiting for the whole reaction, which allows determination of the rate constants of NAD' dissociation from the reaction with Nbs,. Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase shows half-of-the-sites reactivity towards active-site acylation by certain acylating agents. On the other hand, Nbs, is regarded as a full-site reactive reagent, since it reacts very fast with all cysteine-149 and 4mol of Nbs, are essential to inactivate 1 mol enzyme [13]. The pig and rabbit muscle glyceraldehyde-...

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