Subunit interaction in catalysis. Some experimental and theoretical approaches with glyceraldehyde-3-phosphate dehydrogenase.
The evidence for equivalent catalytic sites in tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle has been re-examined and found to be insufficient to exclude alternating or reciprocating sites models. Using a column centrifugation technique, lower limits have been set on the rates of binding and release of coenzyme, and on the ratio of the affinities of NAD+ and NADH. The binding to acyl enzyme has also been examined. The tightly bound NAD+ has been found to be reduced preferentially and kinetically competently when glyceraldehyde 3-phosphate is added, demonstrating the nonequivalence of the sites in the transient reduction of NAD+. The rate of release of the NADH formed rapidly from tightly bound NAD+ was monitored directly by using lactate dehydrogenase and pyruvate to regenerate NAD+. This rate was sufficiently rapid for the NADH formed from tightly bound NAD+ to be a catalytic intermediate. Although these and other results are consistent with a simple alternating sites model, additional approaches appear necessary to find if subunit catalytic cooperativity occurs with this enzyme.
D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D046911:Macromolecular Substances; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding; D011817:Rabbits
D046911:Macromolecular Substances; D009243:NAD; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases