The role of the nicotinamide moiety of NAd+ for negative cooperativity in glyceraldehyde-3-phosphate dehydrogenase as studied by spin-labeled cofactors.

Two derivatives of NAD+ spin-labeled at N6 or C-8 of the adenine ring have been shown previously to be active coenzymes of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). When more than two equivalents of either spin-labeled NAD+ are bound to the tetrameric enzyme, spin-spin interaction is observed in the ESR spectra (Deparade, M.P., Glöggler, K. and Trommer, W.E. (1981) Biochim. Biophys, Acta 659, 422-433). After reduction of enzyme-bound NAD+ spin-labeled at C-8 to the corresponding NADH derivative, the additional peaks due to this spin-spin interaction disappear, which implies that the distance between the two radicals increases. It is proposed that the coenzyme slide further towards the active site upon reduction. ADPR spin-labeled at C-8 binds non-cooperatively, exhibiting a dissociation constant of Kd = 33 microM. Even with 3.5 equivalents bound to the enzyme, spin-spin interaction is not observed. AMP spin-labeled at C-8 combines with two sites per monomer, or a total of eight per tetramer. The respective dissociation constants are Kd1 = 30 microM and Kd2 - 2.3 mM. Phosphate competes with AMP bound to the weak site. Spin-spin interaction is not observed. ATP spin-labeled at C-8 is bound about 10-fold tighter than the corresponding AMP derivative. Four equivalents of ATP are bound per tetramer, but it exhibits no spin-spin interactions. It is concluded that the structure of the pyridine moiety of the coenzymes plays a role in orienting the adenine ring and, thus, affects the cooperativity. The N6 derivative of NAD+ also shows spin-spin interaction; however, only data for the C-8 derivatives are shown in detail.

The synthesis of deuterium-substituted, spin-labeled analogues of AMP and NAD+ and their use in ESR studies of lactate dehydrogenase.

Two spin-labeled analogues of AMP and NAD+ were synthesized, in which a perdeuterated nitroxide radical (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, TEMPAMINE) was attached to C-6 or C-8 position of the adenine ring. The ESR spectra of these derivatives exhibit a 4-fold increase in sensitivity and a concomitant decrease in line-width as compared to the corresponding protonated analogues. The improved resolution of composite spectra consisting of freely tumbling and immobilized components is demonstrated in ternary complexes of the spin-labeled NAD+ derivatives with lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) and oxalate.