Alcohol dehydrogenase data

The Protein Data Bank PDB ID 1A71 Colby T D Bahnson B J Chin J K Klinman J P Goldstein B M Active Site Modifications m a Double Mutant of Liver Alcohol Dehydrogenase Structural Studies of Two Enzyme Ligand Com plexes To be published... 

For a liver alcohol dehydrogenase (LADH) model an NS2O coordination sphere is required. The chelating aldehydes are ideal for the formation of this donor set when combined with bis(pentafluoro-thiophenolato)zinc. Structural data on the complexes with one equivalent of 6-methylpyridine-2-carbaldehyde, 6-methoxypyridine-2-carbaldehyde, 2-(dimethylamino)benzal-dehyde) demonstrate that the coordination sphere for LADH has been reproduced to a close approximation and the corresponding alcohol complexes have also been characterized.354 Other thiophenols have been used to form such complexes but have not been structurally characterized.304... 

Benzyl alcohol can be produced from benzaldehyde (S) by a dehydrogenation reaction catalyzed by yeast alcohol dehydrogenase (YADH). Nikolova et al. (1995) obtained initial-rate data for this reaction using immobilized YADH immersed in iso-octane with 1% v/v water. The following data were obtained ... 

Alberty analyzed the anion effect on pH-rate data. He first considered a one-substrate, one-product enzyme-catalyzed reaction in which all binding interactions were rapid equilibrium phenomena. He obtained rate expressions for effects on F ax and thereby demonstrating how an anion might alter a pH-rate profile. He also considered how anions may act as competitive inhibitors. The effect of anions on alcohol dehydrogenase has also been investigated. Chloride ions appear to affect the on- and off-rate constants for NAD and NADH binding. See also pH Studies Activation Optimum pH... 

Oxidation by direct H transfer from the a-carbon of alcohols to the pyrroloquinoline quinone (PQQ) cofactor of alcohol dehydrogenases was studied using ab initio quantum mechanical methods <2001JCC1732>. Energies and geometries were calculated at the 6-31G(d,p) level of theory, results were compared to available structural and spectroscopic data, and the role of calcium in the enzymatic reaction was explored. Transition state searches at the semi-empirical and STO-3G(d) level of theory provided evidence that direct transfer from the alcohol to C-5 of PQQ is energetically feasible. 

One step or two-step transfer Another major question about dehydrogenases is whether the hydrogen atom that is transferred moves as a hydride ion, as is generally accepted, or as a hydrogen atom with separate transfer of an electron and with an intermediate NAD or NADPH free radical. In one study para-substituted benzaldehydes were reduced with NADH and NAD2H using yeast alcohol dehydrogenase as a catalyst.30 This permitted the application of the Hammett equation (Box 6-C) to the rate data. For a series of benzaldehydes for which o+ varied widely, a value... 

The results of the temperature dependence of the reaction rates of the enantiomers of secondary alcohols with a secondary alcohol dehydrogenase (SADE1) from the thermophilic bacterium Thermoanaerobacter ethanolicus demonstrated a temperature-dependent reversal of stereospecificity (Pham, 1990) (Figure 5.16). At T < 26°C, (S)-2-butanol was a better substrate than (i )-2-butanol on the basis of kCSLt/KM values however, at T> 26°C, (R)-2-butanol was a better substrate than (S)-2-butanol. (S)-2-Pentanol was the preferred substrate at T < 60°C however, the data predict that (i )-2-pentanol would be preferred at T > 70°C. (S)-2-Elexanol was predicted to be the preferred enantiomer only at T > 240°C. Therefore, the concept of isoinversion temperature is as valid for enzyme reactions as for others only the range of catalytically accessible temperatures is smaller. 

Alcohol dehydrogenases can be subdivided with respect to various criteria, two of which are in particular relevant for the application of these enzymes first structural and protein chemical data, especially subunit size and occurrence of metal ions, and second the stereochemical course of the catalyzed reaction and the consequential chirality of the formed alcohol. 

The third subgroup of alcohol dehydrogenases consists of iron-activated enzymes. The first enzyme detected to belong to this group was the ADH II from Zymomonas mobilis [116] followed by the observation that the ADH IV from Saccharomyces cerevisiae shows more than 50% identity to this bacterial ADH [117]. No data about the secondary or tertiary structures of the enzymes in this subgroup are available currently. A prediction based upon the Chou and Fasman analysis [118] indicates that these enzymes are rich in a-helices. 

Experimental data on primary and secondary kinetic isotope effects in the hydride-transfer step in liver alcohol dehydrogenase, LADH, were analyzed using canonical variational transition theory (CVT) for overbarrier dynamics and the optimized multidimentional path (OMT) for the nuclear tunneling (Alhambra et al., 2000 and references therein). This work demonstrates somewhat better agreement of theoretical values of primary and secondary Schaad- Swein exponents calculated by combining CVT/OMT methods with the experimental values instead of CVT and classical transition states (TST). 

Fig. 2. The crystallographic structures of Zn(S-cys)4 cores of (a) aspartate carbamoyltransferase ligated by Cys-Lys-Tyr-Cys and Cys-Pro-Asp-Ser-Asn-Cys (4, 6) and (b) alcohol dehydrogenase ligated by Cys-Gly-Lys-Cys-Arg-Val-Cys (5, 7). These illustrations are based on the X-ray crystallographic atomic coordinates listed in the Protein Data Bank.

The global resolution of heterogeneous decay of fluorescence lifetime data is a powerful technique capable of extracting exponential parameters in such systems as liver alcohol dehydrogenase. The procedure and its sensitivity have been briefly discussed. This methodology represents an important develop-... 

Table I summarizes the sequencing results from alcohol dehydrogenase and the N-terminally blocked glycoprotein ovalbumin. The probable identity of these fragments is indicated. All fragments identified for both proteins by N-terminal sequencing corresponded to cleavage after cystine. The data from ovalbumin are particularly interesting. The structure of ovalbumin is well characterized (5) and contains only one disulfide bond between Cys 73 and Cys 120 yet sequence was obtained following Cys 11 and Cys 30. The bands for these fragments appeared more slowly than the odiers and were fainter in appearance.

The accuracy of the microwave-excitation spectrometric method was verified by comparing results from it with those of atomic absorption analysis for readily available metalloenzymes of known zinc stoichiometry. Carboxypeptidase A (EC 3.4.12.2), carbonic anhydrase (EC 4.2.1.1), alcohol dehydrogenase (EC l.l.l.l), and alkaline phosphatase (EC 3.1.3.1) were dialyzed vs. metal-free bufiFers, then diluted with 10 mmol/ L KCl or 1 mmol/L HCl for metal analysis (24). For atomic absorption analysis, at least lOO- xg samples were required, but microwave excitation required only 0.1 fig. Even though 1000-fold less protein was required for microwave excitation analysis, the agreement between the data obtained by the two methods is excellent (Table II). So little of the reverse transcriptase was available to us that we could not use atomic absorption for its analyses. 

Analytical and enz5onological data lead to the conclusion that zinc is a structural and functional component of ADH, and that it participates in the mechanism of its enzymatic action (Vallee and Hoch, 1955b). The four metal atoms are firmly bound to the protein apoenzyme. Pending the results of investigation in progress, this discussion will assume that the four zinc atoms are bound to the protein in an equivalent manner, and that each atom of zinc acts independently of the other three in the catalytic action of alcohol dehydrogenase. This seems the more acceptable view at present, since Hayes and Velick (1954) found the four binding sites of DPN to be equivalent. 

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