Fluorescence Quenching and Energy Transfer in Complexes between Horse‐Liver Alcohol Dehydrogenase and Coenzymes

Abstract

The fluorescence properties of horse-liver alcohol dehydrogenase were investigated with the aim of separating the contribution of Trp-15 (which is close to the protein surface) from that of Trp-314 (buried in the interior of the protein). Quenching of the protein fluorescence by iodide involves, to a larger degree, the longer wavelength region of the protein emission spectrum and is interpreted to involve only , , at 340 nm and a quantum yield of 0.19). It is shown that quenching by NAD and NADH involves both types of tryptophan, but the 'blue' one to a larger extent. In the case of NADH, radiationless energy transfer between enzyme and reduced nicotinamide ring accounts for less than one half of the total protein fluorescence quenching. Energy transfer between the tryptophan and adenine rings is also possible, but it cannot account for the rest of the protein quenching. Thus, it is suggested that protein conformational changes, following NADH binding, are the cause of part of the fluorescence quenching. The extent to which quenching by NAD can be ascribed to radiationless energy transfer processes is also calculated. It is shown that despite the small spectral overlapping between coenzyme absorption and protein emission, the energy transfer contribution cannot be neglected. However, it is very likely that also in this case a sizeable part of the protein fluorescence quenching comes from protein conformational changes following coenzyme binding. The possible nature of these conformational changes is discussed, taking into account recent X-ray data of enzyme-coenzyme complexes.Horse-liver alcohol dehydrogenase has four tryptophan residues, two per subunit of molecular weight 40000 [l]. Recent X-ray data locate them spatially: Trp-15 is close to the surface and probably exposed to water, while Trp-314 is buried inside the protein, close to the interaction domain of the two subunits property has been used in a number of ways in order to study the interaction between the apoenzyme and coenzyme [4-81. The quenching produced by binding of NADH is usually attributed to energy transfer [5,8] whereas the quenching upon binding of NAD, analogous to the cases of octopine dehydrogenase [9] and yeast alcohol dehydrogenase [I 01, has been ascribed to a protein conformational change [7]. The present work analyzes the overall protein fluorescence in terms of the contribution of the two types of tryptophan residues. It will be shown that it

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

“…Since each of the two enzyme subunits contains one hydrophilic (residue 15) and one hydrophobic (residue 314) tryptophan [16,17], it is possible to state that the longer-wavelength component belongs to Trp-I 5, and the shorter-wavelength one relates to Trp-314. The results obtained are consistent with those of others [8,9,11,12]. Barboy and Feitelson [lo] suggested that 300-nm light excites only Trp-314.…”

“…This finding is not It should be noted that in various works the value of maximal quenching of alcohol dehydrogenase luminescence upon NAD' addition was found to be different. Thus, in [l] it was equal to 20%, in [2] it amounted to 42%, in [5] 47%, in [12] 28%. This is not connected with differences in the experimental conditions.…”

“…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.…”

A study of colchicine tubulin complex by donor quenching of fluorescence energy transfer

“…Fig. 2 shows the emission spectra of tubulin (I), tubulin-colchicine complex (11) and the difference sprectra (111), when excited at 295 nm. The emission maximum of the free protein (I) is at 336.8 nm.…”

Interaction of a Fluorescent Analog of N ‐Deacetyl‐N ‐Methyl‐Colchicine (Colcemid) with Liver Alcohol Dehydrogenase

“…These estimates are significantly shorter than the NADH-TrplS distances, which are 3.6 nm and 5.2 nm, and significantly longer than the NADHTrp314 distances, which are 1.7 nm and 2.4 nm (from X-ray crystal structure data of the NADH-dimethyl sulphoxide-LADH ternary complex ; [I 41) supporting the idea of a binding site distinct from the coenzyme-binding site. Although the specificities of the interaction of colchicine and colcemid with tubulin are compared with that of the enzymesubstrate interaction, it was reported that colchicine and colcemid bind glyceraldehyde-3-phosphate dehydrogenase with Kd values of M and 6X10-'M [18], respectively. Thus, it would be interesting to see whether dehydrogenases possess the general property of binding colchicine and its analogs.…”