Subunit interactions in glyceraldehyde-3-phosphate dehydrogenases. Their involvement in nucleotide binding and cooperativity

Abstract

1. 1. The hybridization of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) was used to study the involvement of subunit interactions in NAD + and NADH binding by these enzymes. 2. 2. In the presence of 1 mM NAD + or NADH no hybrid formation was observed with our preparations of the two enzymes. 3. 3. The inhibition by NADH of the hybrid formation is shown to be a consequence of an unfavourable equilibrium of the hybridization process in the presence of NADH. 4. 4. The inhibition of NAD + of the hybrid formation is shown to be a consequence of both a shift in the equilibrium, as in the case of NADH, and a decrease in the rate of the dissociation of the enzymes. 5. 5. The dimer of the yeast enzyme binds NAD + or NADH with equal affinity irrespective of whether it is combined with another yeast dimer in the yeast tetramer or with a rabbit muscle dimer in the hybrid. 6. 6. The binding of NAD + or NADH to the dimer of the rabbit muscle enzyme is stronger in the rabbit muscle tetramer than in the hybrid; this explains the shift in the equilibrium of the hybridization process caused by these nucleotides. 7. 7. Alkylation of the rabbit muscle enzyme with iodoacetate does not influence the hybridization process in the absence of nucleotides. 8. 8. After alkylation of the rabbit muscle enzyme NADH cannot cause a large shift in the equilibrium of the hybridization process.