On site heterogeneity in sturgeon muscle GPDH: a kinetic approach

• Published in: Biophysical chemistry 1974-02, Vol.1 (3), p.161-174
• Main Authors: Seydoux, Francois, Bernhard, Sidney
• Format: Article
• Language: English
• Subjects: Animals; Apoenzymes; Benzoates; Binding Sites; Computers; Cysteine; Disulfides; Dithiothreitol - pharmacology; Fishes; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; Kinetics; Macromolecular Substances; Models, Chemical; Muscles - enzymology; NAD - pharmacology; Nitro Compounds; Spectrophotometry
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/0301-4622(74)80003-7

The kinetics and stoichiometry of the reaction of sturgeon muscle glyceraldchyde-3-PO 4-dehydrogenase (GPDH) with the disulfide interchange reagent bis(2,2' dithio-bis(5-nitrobenzoate) (DTNB) has been studied in detail. The native enzyme, a tetramer of covalently identical subunits, reacts relatively rapidly with precisely four equivalents of reagent, although there are three cysteine residues per subunit (12 per tetratner). Reaction of these four cystcines leads to total catalytic inactivation; the extent of inactivation is proportional to the fractional reaction. The rate of reaction is dependent on the extent of bound NAD: reactivity being very much greater at unliganded sites. The reaction with apo-enzyme is fastest, bimolecular and monophasic. Over a wide range of NAD concentration, however, the reaction of enzyme with a large molar excess of reagent is precisely biphasic, and each individual kinetic experiment can be analytically described by two pseudo first-order (NAD concentration-dependent) rate constants and two unequal NAD concentration-insensitive amplitudes. The biphasicity in rate is quantitatively explainable on the basis of a C 2 symmetry for the tetrameric subunits with a tighter binding of NAD at two of the four sites, if high reactivity is exclusively dependent on the absence of bound NAD. The inequality in the two amplitudes, however, requires either a more complex or a more dynamic model. Arguments are presented for the appropriateness of a C 2 symmetry model in which intramolecular transconformational isomerization of tight and loose NAD binding sites is possible. The equilibrium constant for the isomerization is estimable from the macroscopic specific rates and amplitudes. This “flip-over” C 2 symmetry model is apropos to all situations of negative cooperativity in ligand binding to tetramers, as is discussed.