Luminescence studies on the conformational behavior of horse-liver alcohol dehydrogenase

Abstract

The luminescence quenching and conformational behaviour of alcohol dehydrogenase from horse liver upon substrate binding has been studied. It was shown that the binding of NADH and NAD+ to the enzyme resulted in the quenching of Trp-314 luminescence, whereas the luminescence of Trp-15 was not quenched. In this case nonradiating energy transfer from Trp-314 to NADH was observed. An essential energy transfer from Trp-15 to NADH and between the two Trp-314 residues of both subunits of the enzyme was not revealed. The quenching of the enzyme luminescence upon NAD+ binding was caused mainly, by NAD' reduction to NADH. It was assumed that the release of the proton upon NAD' binding occurred due to the reduction. Binding of ethanol, ADP or adenosine did not result in essential conformational changes of the enzyme.The origin of the luminescence of alcohol dehydrogenase from horse liver and its quenching by various substrates has been studied in detail [l -141. Nevertheless the reasons for the quenching of this luminescence by pyridine nucleotides and adenine compounds have not been completely elucidated. It was found that in the enzyme-NADH complex the quenching was due to energy transfer [4], but some authors [1,2,5,12] showed a quenching effect of NAD', which has no absorption 0.4-cm and 1 -cm quartz cuvettes, respectively. The spectra were corrected for screening and reabsorption; the spectral width of the monochromator slits was not more than 4 nm. The lifetime of tryptophanyl residues were measured with an SLM-4800 spectrometer as described in [18]. All measurements were performed at 23 "C.RESULTS AND DISCUSSION band at 340 nm, which suggests there is no energy transfer. Thus, it was assumed that NAD' binding results in conformational changes in the enzyme. The quenching observed was explained by the change in the microenvironments of tryptophan residues. On the other hand, it was found calorimetrically that the enthalpy of binding of pyridine nucleotides with alcohol dehydrogenase was near zero [15]. This is indirect evidence for the fact that the conformational changes are not strong. The direct data on X-ray analysis showed that the conformation of enzyme-substrate complexes is very much like that of the apoenzyme [16,17]. The present work aims to elucidate the origin of alcohol dehydrogenase luminescence quenching by pyridine nucleotides. MATERIALS AND METHODSPreparations of alcohol dehydrogenase from horse liver (Reanal), stored at 0 "C, was dialyzed and added immediately before the experiment to 10 mM Tris buffer, pH 7.5, to a final concentration of not more than 0.14 mg protein/ml buffer. Solutions of reduced nicotinamide -adenine dinucleotide (Reanal), oxidized nicotinamide -adenine dinucleotide (Reanal), adenosine diphosphate (Serva), adenosine (Reanal) and acetaldehyde (Reachim) were prepared immediately before the experiment in double-distilled water.The excitation and emission spectra were recorded with Hitachi MPF-44B and SLM-4800 spectrofluorimeters in Enzyme. Alcohol dehydrogenase (EC 3.1.1.1).The sp...

Tryptophan luminescence from liver alcohol dehydrogenase in its complexes with coenzyme. A comparative study of protein conformation in solution

“…The closer proximity of NMN to Trp-314 explains also the preferential quenching of this residue over the distal (27 Á) Trp-15. The mechanism of quenching by NAD+, on the other hand, is still debated, and although several proposals have been advanced in the literature, none is really satisfactory (Subramanian et al, 1981;Eftink & Selvidge, 1982;Vekshin, 1984).…”

Comments on ‘Luminescence studies on the conformational behavior of horse‐liver alcohol dehydrogenase’, by N. L. Vekshin

“…In [1] it has been shown that without dialysis of horseliver alcohol dehydrogenase, about 55% quenching takes place upon NAD' addition. This is mainly due to reduction of NAD+ to NADH.…”