Pig muscle 3-phosphoglycerate kinase was complexed with I-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used.By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (& < 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 "C. (EC 1.2.1.22).binding sites or to the existence of regulatory sites [5, 8, 161. Equilibrium binding studies seem to support the hypothesis of a secondary binding site, especially in the case of nucleotide substrates [lo, 12, 141. However, nuclear magnetic resonance [12,18], spectrophotometric [15] and X-ray diffraction [19-211 studies indicate one binding site for the nucleotide and one site for 3-phosphoglycerate.To explain the observed anomalies, Scopes [I41 has recently suggested a mechanism with a single site for each substrate, in which MgATP and 3-phosphoglycerate accelerate the two-step release of 1,3-bisphosphoglycerate, which is assumed to be the rate-limiting step in catalysis. Moreover, in another inhibition study on 3-phosphoglycerate kinase [22] the alternative possibility of a steady-state random mechanism was raised.In the present paper the possible mutual influence of substrates in their binding to 3-phosphoglycerate kinase was investigated by the aid of the fluorescence signal of l-anilino-8-naphthalenesulfonate (ANS) bound to the pig muscle enzyme.ANS, the extensively used fluorescent label which binds reversibly to different proteins (cf.[23] and references therein), was used to investigate several nucleotide-binding enzymes. The binding of ANS has often been traced to the hydrophobic adenine-binding pocket of various dehydrogenases [24-271, whereas non-competitive binding with respect to the nucleotides was observed in other cases [28, 291. In addition to the substrate binding studies, steady-state kinetic studies of the enzymic reaction were performed under the same conditions on pig muscle 3-phosphoglycerate kinase.

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Maleylation of about four groups per tetramer of NAD-free glyceraldehyde-3-phosphate dehydrogenase or of enzyme -NAD, complex inactivates the enzyme. However, enzyme -NAD, complex is inactivated only by maleylation of about 24 groups per tetramer. This shows that the presence of two moles of the relatively loosely bound coenzyme in addition to the firmly bound ones protects the tetramer against inactivation. Up to the maleybtion of about 12 groups per tetramer NAD-free enzyme, the reactive SH-groups do not react with maleic anhydride. Maleylation of the enzyme -NAD, complex up to the same extent does not affect the Racker band characteristic of the enzyme -coenzyme complex and the coenzyme remains firmly bound to the enzyme. However, after maleylation of NAD-free tetrameric enzyme only two NAD molecules can be rebound to the inactive enzyme.Four moles of ATP can be bound tightly to the NAD-free enzyme. Two of them can be bound within a few minutes, whereas the further two only after prolonged incubation. Binding of two moles of ATP to the NAD-free tetrameric enzyme in the absence of 2-mercaptoethanol results in the irreversible inactivation of the enzyme. I n the presence of 2-mercaptoethanol the inhibition is reversible and of mixed type between ATP and NAD.The above data provide evidence for the functional non-identity and interaction of subunits. The dissociation of tetrameric enzyme is increased by ATP or maleic anhydride and this process is irreversible in the absence of 2-mercaptoethanol. An enzymically active dimeric form of the ATP-treated enzyme has been isolated in the presence of 2-mercaptoethanol. ATP treatment causes only a slight change in the steric structure of the protein as measured by difference spectrophotometry, showing that the dissociation into dimers is not accompanied necessarily by the unfolding of the molecule.Interaction was observed between active and inactive enzyme molecules, which suggests the dissociation of oligomer and recombination of subunits.I n solution, the enzyme exists as a tetramer-dimer equilibrium mixture and both forms are enzymically active.Glyceraldehyde-3-phosphate dehydrogenase isolated from mammalian muscle or from yeast is a tetramer [1,2]. It is known that glyceraldehyde-3-phosphate dehydrogenase isolated from different phyla or classes, e.g. mammals, yeast, fishes, etc. are different [3 -81. However, the enzymes isolated from different mammalian muscles (different species : rabbit, swine, ox, ete.) are completely identical enzymically, physico-chemically, immunologically and in even their complete amino acid sequence [3,4,8 to 151. Therefore the literature data on enzymes from different mammalian muscles will be treated uniformly.Enzymes. ~-Glyceraldehyde-3-phosphate dehydrogenase or ~-glyceraldehyde-3-phosphate : NAD oxidoreductase, phosphorylating (EC 1.2.1.12) ; a-glycerophosphate dehydrogenase or ~-glycerol-3-phosphate : NAD oxidoreductase (EC 1.1.1.8).The subunits of the mammalian enzymes are chemically identical [lo]. Earlier data have suggested that ...

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