title: Conformational motility in D-glyceraldehyde-3-phosphate dehydrogenase influenced by subunit interactions.
abstract: Binding of four molecules of NAD to pig muscle glyceraldehyde-3-phosphate dehydrogenase decreases the apparent reactivity of Cys-153 -- a residue exposed only temporarily -- towards PMB in all four subunits of the enzyme. However, the change of reactivity is not a linear function of the degree of saturation with coenzyme, inasmuch as the first two, tightly bound NAD's exert a much larger effect than do the other two. The apparent reactivity of Cys-153 was investigated in GAPD's produced by hybridization of enzymes modified on residue Cys-149 with different reagents. The homotetramer-NAD complexes of these modified species were shown to exhibit different dissociation constants: native GAPD less than (alkylated--Cys-149)-GAPD less than (mercaptidated--Cys-149)-GAPD. The tight binding of 2 NAD's on the hybrid tetramers decreases the reactivity to the same extent in liganded and non-liganded subunits. The decrease in the apparent reactivity of Cys-153 is due to the restriction of local conformational motility around thes residue. Binding of NAD shifts the equilibrium towards a more closed form of the protein, whereas the rate constant of mercaptide formation itself remains unaltered. These findings suggest that the NAD-induced conformational changes are also reflected in the local fluctuation of the protein around Cys-153. The subunit interactions still operate in the hybrids and mediate the NAD-induced conformational changes.
mesh_terms: D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D005033:Ethylmaleimide / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008626:Mercuribenzoates / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D009243:NAD; D011485:Protein Binding; D011487:Protein Conformation; D013552:Swine
chemical_list: D046911:Macromolecular Substances; D008626:Mercuribenzoates; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine; D005033:Ethylmaleimide