Conformations of Diphosphopyridine Coenzymes upon Binding to Dehydrogenases

Abstract

The binding of oxidized as well as reduced coenzyme to some dehydrogenases has been studied under different concentration ratios and temperatures by nuclear magnetic resonance spectroscopy. A significant difference in the spectral behavior between DPN+ and DPNH upon binding is interpreted in terms of fast and slow on-off rates relative to the nuclear magnetic resonance time scale in the binding of these two coenzymes. Significant downfield shifts of DPN+ were observed upon binding, comparable in magnitude to those expected upon opening (destacking) of the coenzymes in the case of chicken-muscle and lobster-tail lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1). A preliminary survey of several other dehydrogenases is consistent with these findings. In the case of 3-phosphoglyceraldehyde dehydrogenase, there is a possibility that the coenzyme exists in the folded form.Nuclear magnetic resonance spectroscopy (NMR) is now extensively applied to probe the active sites of enzymes in solution (1, 2). Most of these studies have been concerned with reversible enzyme-inhibitor or substrate interactions (3-8).Information about the active sites is often inferred from the effects that the enzyme has on the chemical shifts (9, 10), and line-widths (11), as well as on relaxation times (12) of specific resonances of the ligand.It has been shown that in neutral aqueous solution the pyridine coenzymes, DPN+ and DPNH, exist in rapid equilibrium between open and folded conformations (13-15). Investigations regarding the actual conformations of these coenzymes in the active sites of dehydrogenases showed discrepancies depending on the methodology used. The fluorescence energy-transfer study of Velick indicates that the reduced coenzyme appears to exist in an open conformation upon binding to most dehydrogenases; 3-phosphoglyceraldehyde dehydrogenase being the only exception (16,17). From a 5A x-ray map, Adams et al. (18) 1593 the reduced coenzyme exists in an intramolecular folded conformation in the active site of this enzyme (19). The observation of the upfield shift of both H8 and H2 protons on the adenine moiety of DPNH in the presence of this enzyme led to the above conclusions.We have recently studied again the binding of pyridine coenzymes to various dehydrogenases under various experimental conditions. From the results of our detailed study, we have been able to ascertain the conformation of coenzymes when bound to dehydrogenases in solution. MATERIALS AND METHODSAlcohol dehydrogenase from yeast (YADH) was purchased from both Calbiochem, Calif. and Sigma Chemical Corp., Mo. Lactate dehydrogenase from chicken muscle (M4LDH) and chicken heart (H4LDH) were prepared in our laboratory by Mr. Francis Stolzenbach (20). Lactate dehydrogenase from lobster tail was isolated and purified as reported elsewhere (21,22). The specific activities of dehydrogenases used in this study were measured before each experiment.

Intra molecular Hydrogen Bonding in Flavin Adenine Dinucleotide

“…A recent report from our laboratory has described the configuration that NAD+ assumes upon binding to any of a number of dehydrogenases (21). In nearly all cases, evidence was obtained consistent with the view that destacking of NAD+ accompanies binding at the coenzyme site.…”

Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

“…These results broadly support the view, originally put forward by Velick (1961), that the mode of binding of NAD+ to glyceraldehyde 3-phosphate dehydrogenase differs considerably from its mode of binding to lactate and malate dehydrogenases (see, e.g., the recent n.m.r. studies by Lee et al, 1973).…”

Potential Dependence of the Conformations of Nicotinamide Adenine Dinucleotide on Gold Electrode Determined by FT−Near-IR−SERS

“…A number of studies have shown that this coenzyme mainly assumes a stacked conformation in solution. − In contrast, X-ray diffraction analysis has indicated an extended NAD conformation when bound in biochemical systems. ,, Knowledge of the factors influencing the folding process would be desirable and could lead to an increased understanding of the role of this cofactor in enzymatic redox reactions.…”

“…Various techniques have been used to identify the conformations assumed by NAD in different environments such as aqueous solution, − binding to enzymes or metal ions, ,, crystallization, , or adsorption on metal surface. − Because a charged metal surface may serve to mimic a biological interface and provide informatioin about the electrochemistry of the bound NAD, there is considerable interest in studies of such models. However, most of such measurements reported previously were performed under ex-situ conditions, and the proposed conformation models were usually based upon indirect evidence.…”