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)

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.
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PMID:Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase. 675 25

When RNA isolated from the Drosophila melanogaster alcohol dehydrogenase (ADH) negative mutant CyOnB was translated "in vitro" in the presence of yeast opal suppressor tRNA, a wild type size ADH protein was obtained in addition to the mutant gene product. This identifies the CyOnB mutant as an opal (UGA) nonsense mutant. From the molecular weight of the mutant protein, and from the known sequence of the ADH gene (Benyajati et al., Proc.Natl.Acad.Sci. USA 78, 2717-2721, 1981), we conclude that the tryptophan codon UGG in position 234 has been changed into a UGA nonsense codon in the CyOnB mutant. Furthermore, we show that the UAA stop codon of the wild type ADH gene is resistant to suppression by a yeast ochre suppressor tRNA. This is in contrast to the high efficiency of suppression of the CyOnB UGA nonsense codon, despite an almost identical codon context.
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PMID:In vitro suppression of a nonsense mutant of Drosophila melanogaster. 681 27

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.
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PMID:Rotational freedom of tryptophan residues in proteins and peptides. 684 81

Protease B [EC 3.4.22.9] was purified from baker's yeast by plasmolysis of yeast, acid activation, acid precipitation, and column chromatographies on QAE-Sephadex, SP-Sephadex, D-tryptophan methyl ester-Sepharose 4B and Sephadex G-100. The purified enzyme was inhibited by phenylmethylsulfonyl fluoride and sulfhydryl-blocking reagents. Chymostatin and antipain at extremely low concentrations (1 micro M) inhibited the protease B. The effects of the enzyme on various yeast enzymes were examined by measuring their inactivation. The enzyme inactivated 6-phosphogluconate dehydrogenase [EC 1.1.1.44] and uricase [EC 1.7.3.3], but not malate dehydrogenase [EC 1.1.1.37], alcohol dehydrogenase [EC 1.1.1.1], glutamate dehydrogenase [EC 1.4.1.3], glucose-6-phosphate dehydrogenase [EC 1.1.1.49] or hexokinase [EC 2.7.1.1].
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PMID:Purification and characterization of yeast protease B. 699 57

The tryptophan fluorescence decay of horse liver alcohol dehydrogenase, at 10 degrees C in 0.1 M pH 7.4 sodium phosphate buffer, with excitation at 295 nm, is a double exponential with time constants of 3.8 and 7.2 ns. Within experimental error, the two lifetimes remain constant across the emission spectrum. Only the 3.8-ns lifetime is quenched in the NAD+-pyrazole ternary complex, and only the 7.2-ns lifetime is quenched by 0-0.05 M KI. On the basis of these results, we assign the 3.8-ns lifetime to the buried tryptophan, Trp-314, and the 7.2-ns lifetime to the exposed tryptophan, Trp-15. The steady-state lifetime-resolved emission spectrum of Trp-15 has a maximum at approximately 340 nm and that of Trp-15 is at approximately 325 nm. The total time-resolved emission, after 40 ns of decay, has a maximum between 338 and 340 nm and is primarily due to the Trp-15 emission. As a consequence of the wavelength dependence of the preexponential weighting factors, there is an increase in the average lifetime from the blue to the red edge of the emission. This increase reflects the change in the spectral contributions of Trp-15 and Trp-314. Consideration of the spectral overlap between the emission spectra of the two tryptophans and the absorption due to formation of the ternary complex, as well as the distances between the two residues and the bound NAD+, shows that the selective fluorescence quenching in the ternary complex can be accounted for entirely by singlet-single energy transfer. The decay of the fluorescence anisotropy was measured as a function of temperature from 10 to 40 degrees C and is well described by a monoexponential decay law. Over this temperature range the calculated hydrodynamic radius increases from 33.5 to 35.1 A. Evidently, the indole groups of Trp-15 and Trp-314 rotate with the protein as a whole, and there is some expansion of the protein matrix as the ambient temperature is increased.
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PMID:Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase. 702 98

Selection of petite strains of yeast (that is, strains unable to respire aerobically) on media containing allyl alcohol will result in enrichment for mutants at the ADC1 locus. This locus codes for the constitutive alcohol dehydrogenase, ADH-I, which is primarily responsible for the production of ethanol in yeast. The mutant enzymes are functional, and confer resistance to allyl alcohol on the cell by shifting the NAD-NADH balance in the direction of NADH. These mutants exhibit altered Km's for cofactor, substrate, or both, and often have altered Vmax's. In this paper, the methodology for obtaining these mutants and for determining the amino acid substitutions responsible for these changes is presented. Several new mutants have been at least approximately localized, and one, DB-AA3-N15, has been shown to be due to the substitution of an arginine for a tryptophan at position 54. This substitution would be expected, by analogy with the known tertiary structure of the horse liver alcohol dehydrogenase, to decrease the hydrophobic environment of the active site pocket. The substitution has a pronounced effect on the Km for ethanol, but far less on that for acetaldehyde. The current status of investigation of other classes of functional mutants of this enzyme, and the potential both for selection of useful variants of this molecule and for an increase understanding of its function are discussed.
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PMID:Functional mutants of yeast alcohol dehydrogenase. 703

We used phase fluorometry to investigate the wavelength dependence of the fluorescence lifetimes of N-acetyl-L-tryptophanamide (NATA) in solvents of varying viscosity and the lifetimes of tryptophan in human serum albumin, melittin, and liver alcohol dehydrogenase. In highly fluid solvents, and in completely vitrified solvents, the lifetime of NATA was constant across its emission spectrum. In viscous solvents, such as propylene glycol at -9 degrees C, the lifetimes of NATA increased across its emission spectrum, with the values being 3.3, 5.5, and 8.1 ns at 317, 344, and 400 nm, respectively. These wavelength-dependent lifetimes appear to be a result of reorientations of solvent dipoles around the excited state dipole moment of the indole moiety. For the three proteins investigated, the fluorescence lifetimes of tryptophan increased with increasing wavelength in a manner comparable to that observed for NATA in propylene glycol. These observations indicate that these protein matrices can reorientation around their tryptophan residues on the nanosecond timescale, and illustrate the potential of phase fluorometry for quantifying the details of these dipolar relaxation processes.
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PMID:Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence. 735 62

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.
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PMID:Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans. 781 33

Circularly polarized luminescence (CPL) spectroscopy provides information on the excited-state chirality of a lumiphore analogous but complementary to information regarding the ground-state chirality derived from circular dichroism. The sensitivity of CPL spectra to molecular conformation makes this technique uniquely suited for the study of biomolecular structure, as extensively demonstrated in earlier studies. Unfortunately, the CPL spectra of many biomolecules often contain significantly overlapping contributions from emitting species either because multiple lumiphores are present (e.g., tryptophan residues in a protein) or because multiple conformations of the biomolecule simultaneously exist, each with a unique CPL spectrum. Increased resolution between individual contributions to the CPL may be achieved by time-resolving this signal, thus taking advantage of the fact that, as a rule, each of the emitting species also has a characteristic decay time associated with its electronically excited state. In addition, the time resolution provides information regarding dynamics associated with the different chiral states of the system. The present study describes an instrument for the determination of time-resolved CPL (TR-CPL) with subnanosecond resolution and its application to several chiral systems. The technique was first demonstrated on a model system with a strong time-dependent CPL signal. Subsequently, the circularly polarized component in the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) bound to liver alcohol dehydrogenase was time-resolved. The CPL of NADH in the binary enzyme-coenzyme complex is time-dependent, reflecting structural differences around the reduced nicotinamide possibly due to a dynamic restructuring. In contrast, the CPL of the coenzyme in the ternary complex formed with enzyme and the substrate analog isobutyramide is essentially time-independent, likely reflecting a more rigid binding domain. Since the linear polarization of the fluorescence of the two complexes did not show any local flexibility of the NADH chromophore, the excited-state conformational rearrangement of the binary complex indicates a subtle change in its interactions with group(s) in direct contact with it.
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PMID:Nanosecond time-resolved circular polarization of fluorescence: study of NADH bound to horse liver alcohol dehydrogenase. 783 31

Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants.
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PMID:Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii. 834 50


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