Synthesis of spin-labeled photoaffinity derivatives of NAD+ and their interaction with lactate dehydrogenase.

The synthesis of NAD+ derivatives spin-labeled at either N6 or C8 of the adenine ring is described, in which the carboxamide function of the nicotinamide moiety is replaced by a diazirine ring. Irradiation of these compounds at 350 nm generates a carbene which will react with any functional group in its vicinity including hydrocarbons. Both NAD+ derivatives form tight ternary complexes with lactate dehydrogenase and were covalently incorporated into this enzyme. They may be employed for ESR studies when non-covalent interactions are too weak for motionally restricted species to be observed.

Cyanylation of 3-hydroxybutyrate dehydrogenase. The "essential" sulfhydryl group is not involved in catalysis.

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with N-ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [McIntyre, J. O., Fleer, E. A. M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3'-dithiobis(6-nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited approximately 50% in excess substrate. Derivatization of the reactive sulfhydryl group results in loss of this substrate inhibition, the modified enzyme being at least three-fold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the S-cyanylated and S-(carboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of 100mM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH-monomethylmalonate) (spin-labeled NAD-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism.

Complex formation between nucleotides and D-beta-hydroxybutyrate dehydrogenase studied by fluorescence and EPR spectroscopy.

D-beta-Hydroxybutyrate dehydrogenase (D-3-hydroxybutyrate:NAD+ oxidoreductase, EC 1.1.1.30) is a lipid-requiring enzyme which specifically requires phosphosphatidylcholine for enzymic activity. The phosphatidylcholine modifies the binding and orientation of the coenzyme, NAD(H), with respect to the enzyme. In the present study, two derivatives of NAD, spin-labeled either at N-6 or C-8 of the adenine ring, were found to be active as coenzyme. The binding affinity of NADH to the enzyme was opitimized by increasing the salt concentration and increasing the pH from 6 to 8, with the pK at 6.8. Monomethylmalonate, a substrate analogue, was found to enhance NADH binding (Kd is reduced from 4 to 1 microM). Sulfite strongly enhances the binding of NAD+ via the enzyme-catalyzed formation of an adduct of sulfite with the nucleotide; the Kd for binding of NAD-sulfite is in the micromolar range, whereas NAD+ binding is more than a magnitude weaker. The binding of spin-labeled NAD(H) was further characterized by EPR spectroscopy. Increased sensitivity and resolution were obtained with the use of NAD(H) analogues perdeuterated in the spin-label moiety. For these analogues bound to D-beta-hydroxybutyrate dehydrogenase in phospholipid vesicles, EPR studies showed the spin-label moiety to be constrained and revealed two distinct components. Increasing the viscosity of the medium by addition of glycerol affected the EPR spectral characteristics of only the component with the smaller resolved averaged hyperfine splitting. The stage is now set to study motional characteristics of the enzyme, using these spin-labeled probes which mimic the coenzyme.

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.