Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.1 (alcohol dehydrogenase)
9,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.
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PMID:pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases. 2 33

The techniques of fluorescence enhancement, fluorescence quenching, fluorescence polarization, and equilibrium dialysis are utilized to study the binding properties of coenzyme to horse liver alcohol dehydrogenase. Polarization of fluorescence and equilibrium dialysis show that NADH binds to alcohol dehydrogenase with a stoichiometry of 6 mol per mol of enzyme, in contrast to the value of 2 determined from fluorescence enhancement measurements. NAD+ also binds with a stoichiometry of six as was determined by equilibrium dialysis. The two NADH sites which bind coenzyme more tightly and which are revealed by fluorescence enhancement measurements are designated the catalytic sites. Binding of coenzyme to the four ancillary sites does not alter the quantum yield of NADH but results in a 20% contribution to quenching of enzyme's tryptophan fluorescence. From the emission anisotropy of bound NADH of 24.0% for the additional sites and 28.1% for the catalytic sites and their relative fluorescence lifetimes at the same wavelengths of excitation and emmision, we conclude that the nicotinamide ring of NADH bound to the additional sites exhibits a freedom of motion independent of the macromolecule, while that bound to the catalytic sites is more rigidly held. Polarization of fluorescence yields negative intrinsic free energies of 9.2 and 7.5 Cal M-1 for NADH interaction with the catalytic and additional sites, respectively. Although these values are 1.3 to 2.0 Cal higher than those determined by fluorescence quenching and equilibrium dialysis, the mean Hill coefficient of 1.76 plus or minus 0.06, the titration span of 2.4 logarithmic units and coupling free energies (in magnitude and sign) are the same for all these techniques. The above difference in the intrinsic free energies are attributed largely to the different modes of interaction of excited and unexcited NADH molecules with alcohol dehydrogenase.
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PMID:Coenzyme interaction with horse liver alcohol dehydrogenase. Evidence for allosteric coenzyme binding sites from thermodynamic equilibrium studies. 16 20

Extensive and informative steady-state kinetic investigations of the mechanisms of horse liver alcohol dehydrogenase have recently been complemented by observations of the fluorescence and spectroscopic characteristics of transient intermediates by rapid-reaction techniques. In this way it was possible to study separately steps involved during enzyme-substrate complex formation and during the catalytic process. It can be shown that a proton is liberated during complex formation before the transfer of a hydride ion from ethanol to form NADH. This must be due to a change in pK of a group on the enzyme protein and is linked to a change in tryptophan fluorescence. Pressure relaxation techniques have enabled us to study the rate constants of the change in tryptophan fluorescence linked to NAD+ binding and proton dissociation. We have shown that NAD+ binding occurs in two steps: a rapid secondorder association, followed by the substrate-induced isomerization to form the reactive enzyme-substrate intermediate. The isomerization rate constants were determined in both directions and their role in the overall reaction mechanism could be identified.
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PMID:The consequences of nucleotide binding to liver alcohol dehydrogenase. 21 95

Some physical and chemical properties of the monomeric NADP+-dependent aldehyde reductase (previously called TPN-L-hexonate dehydrogenase or D-glucuronate reductase) from pig kidney have been examined. The amino acid composition has been determined. Four of the five thiol groups react with p-mercuribenzoate at pH 7, with no resulting loss of catalytic activity. High concentrations of p-mercuribenzoate cause complete enzyme inhibition, which can be partly reversed by addition of aldehyde reductase is low (9%, estimated from the ellipticity at 208 nm), and 70 to 80% of the tyrosine and tryptophan residues aare buried within the molecule. One molecule of NADPH binds to the enzyme (Kp equal 25 muM), causing a blue shift and enhancement of the coenzyme fluorescence, and suggesting that the environment of the active site is hydrophobic. In the reduction of D-glyceraldehyde, catalyzed by aldehyde reductase, the pro-4R "A" hydrogen of NADPH attacks the re face of the carbonyl group. This stereospecificity is the same as in the reductions of D-glyceraldehyde and acetaldehyde effected by rabbit muscle dehydrogenase and liver alcohol dehydrogenase, respectively.
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PMID:Properties of the nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductase from pig kidney. Amino acid composition, reactivity of cysteinyl residues, and stereochemistry of D-glyceraldehyde reduction. 23 31

The detergents which contain a hydrocarbon side chain longer than 16 cabron atoms were used as a perturbant for the study of protein structure. ta low concentration of cetyldimethylbenzylammonium chloride (CDBA) caused difference spectra for Ac-Trp-OEt and AC-Tyr-OEt. The delta e values at their difference maxima became constant above 30 mM of cetyldimethylbenzylammonium chloride, 1430 at 294 nm for Ac-Trp-OEt and 450 at 288 nm for Ac-Tyr-OEt. These delta e values are higher than any other delta e values resulting from solvent effects by such a remarkably low concentration of organic reagents described in the literature so far. The absence of denaturation blue shift in the difference spectra and the fact that the optical rotatory dispersion of the proteins examined in the present study was not changed significantly by cetyldimethylbenzylammonium chloride indicate that the secondary and tertiary structures of the proteins were not destroyed by cetyldimethylbenzylammonium chloride. These characteristics, together with small overlapping of their difference spectra at 288 and 294 nm were advantageous in the determination of tryptophan and tyrosine residues exposed in glucagon, insulin and alcohol dehydrogenase from yeast. No tyrosine residues in ribonuclease A was accessible to cetyldimethylbenzylammonium chloride. Unusual difference spectrum with a peak at 298 nm was observed for lysozyme which is known to contain tryptophan residues in special environments. Ovalbumin gave a novel unusual difference spectrum with a peak at 290 nm and a shoulder at 298 nm, showing the existence of unusual tryptophan and probably tyrosine residues in the molecule.
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PMID:Interaction between proteins and detergents which contain a hydrocarbon chain longer than 16 carbon atoms. II. Difference spectra of various proteins in cetyldimethyl-benzylammonium chloride. 23 67

Pulse radiolysis has been used to investigate the rates and transient spectra for the reactions of free radicals with beef heart lactate dehydrogenase at pH 7. Analysis of the results leads to second-order rate-constants for eaq-, .OH, .I, .Br2-, .I2- and .(CNS)2- which are, respectively, 24, 21, 10, 0.55, 0.43 and 0.15 in units of 10(10) M-1 s-1 with uncertainties of +/- 20 per cent. Those for .I and .I2- are similar to the corresponding rate-constants for the related enzyme alcohol dehydrogenase. The spectra of the transient species produced by .OH, .Br2- and .(CNS)2- all showed evidence for reactions with tyrosine and tryptophan residues, and in general terms the magnitudes of the rate-constants appeared to increase with the oxidizing abilities of the radicals. The implication of the results for understanding the mechanism of deactivation by free radicals is discussed.
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PMID:Pulse radiolysis of lactate dehydrogenase. 30 25

When tryptophyl side chains are hidden within relatively inflexible domains of globular proteins, the lifetime of the phosphorescence from these residues provides a measure of the local conformational flexibility. The phosphorescence decay from the tryptophan buried at the base of the nucleotide-binding domain in liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was monitored between 1 and 40 degrees C to determine the energetics associated with the rate of local unfolding. The slow rate at which this process takes place is found to result from a high entropic barrier rather than from the disruption of strong intramolecular interactions. This observation along with the response of the system to solvent perturbations points to the significance of solvent-protein interactions in determining conformational flexibility.
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PMID:Phosphorescence evidence for the role of solvent--protein interactions in the energetics of conformational flexibility of liver alcohol dehydrogenase. 39 11

Difference fluorescence emission spectra, reciprocal Stern-Volmer plots, and variable excitation wave-lengths have been used to evaluate the selective quenching of the two tryptophan residues/subunit of liver alcohol dehydrogenase. Trp-15, at the surface of the enzyme, is quenched by KI consistent with a collisional mechanism, and has a blue-shifted excitation and red-shifted emission spectrum when compared with the spectral properties of TRP-314, which is in a hydrophobic milieu at the subunit interface of the dimeric enzyme. With excitation at 295 nm, Trp-314 is 80% quenched by formation of a ternary enzyme.NAD+.trifluoroethanol complex, and the quenching is essentially additive to that caused by KI. Alkaline pH also results in selective quenching of Trp-314. These results, and considerations of the three-dimensional structure of the enzyme, indicate that the quenching of protein fluorescence of liver alcohol dehydrogenase by either ternary complex formation or alkaline pH is due to resonance energy transfer to tyrosinate. Likely candidates as energy acceptors are the Tyr-286 residues are within transfer distance for each Trp-314 residue, as well as being at the surface of the enzyme and 30 A from the active center zinc atom. Alkaline pH directly ionizes this tyrosine residue, while ternary complex formation causes a conformational change resulting in its ionization.
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PMID:The mechanism of quenching of liver alcohol dehydrogenase fluorescence due to ternary complex formation. 71 68

N-Acetyltryptophanamide (NATA), when illuminated anywhere within the 280-nm absorption band, has an emission lifetime of 3.1 ns. The tryptophan residues in liver alcohol dehydrogenase (LADH), however, when excited at 280 nm exhibit two lifetimes of r1 = 2.2 and of r2 = 5.7 ns. Excitation at 300 nm yields a single decay of 5.0 ns. It is shown that at the latter wavelength, only the two (equivalent) tryptophan residues buried within the LADH structure are excited. The reaction rate of the NATA fluorescence quenching by ionic and nonionic quenchers is practically independent of the temperature (between 5 and 41 degrees C). The same substances were used to quench the tryptophan fluorescence in LADH. Here (in the same temperature range), the quenching rate decreases drastically with a decrease in temperature. These findings are discussed in terms of conformational fluctuations in LADH, whereby the temporal movement of the polypeptide chains opens channels through which the above quencher molecules can diffuse and reach the tryptophan residues located within the enzyme structure.
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PMID:Fluorescence quenching as an indicator for structural fluctuations in liver alcohol dehydrogenase. 71 66

The intrinsic fluorescence lifetimes of horse liver alcohol dehydrogenase (EC 1.1.1.1) and pig heart isocitrate dehydrogenase (EC 1.1.1.42) have been determined to be 5.36 ns and 4.84 ns, respectively. When reduced coenzyme is bound, the fluorescence lifetime of alcohol dehydrogenase is reduced to 4.98 ns while that of isocitrate dehydrogenase remains unchanged. Oxidized coenzymes have no effect on fluorescence lifetimes of alcohol and isocitrate dehydrogenases. This virtual constancy of protein fluorescence lifetimes has allowed the conclusion to be reached that in protein-ligand complexes with equilibrium constants in the range of 10(4)-10(6) M(-1), the static mode of quenching is substantial. The observation of resonance energy transfer in alcohol dehydrogenase-NADH complex facilitates the determination of the distance between tryptophan and the reduced nicotinamide ring involved in the transfer as 30.6 A, compared to the effective molecular radius of 36.2 A for alcohol dehydrogenase. The increased rotational relaxation times of coenzyme-bound alcohol dehydrogenase relative to the unliganded form (sigmah = 72 ns) indicate in this protein structural fluctuations occurring in the time range of nanoseconds.
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PMID:Protein fluorescence and electronic energy transfer in the determination of molecular dimensions and rotational relaxation times of native and coenzyme-bound horse liver alcohol dehydrogenase. 97 11


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