Purification and properties of alcohol dehydrogenase from a mutant strain of Candida guilliermondii deficient in one form of the enzyme

1992 ◽  
Vol 38 (9) ◽  
pp. 953-957 ◽  
Author(s):  
Retno Indrati ◽  
Yoshiyuki Ohta
Keyword(s):  
Alcohol Dehydrogenase ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Candida Guilliermondii ◽  
Secondary Alcohols ◽  
Primary Alcohols ◽  
Subunit Molecular Weight ◽  
Purification And Properties ◽  
A Subunit ◽  
Better Than

Alcohol dehydrogenase (ADH1) was purified from Candida guilliermondii strain B10-05 to homogeneity, using affinity chromatography on triazine dyes and gel filtration. The enzyme was tetrameric, with a subunit molecular weight of 38 000. The purified enzyme oxidized primary and secondary alcohols, although it preferred primary alcohols. Its activity toward secondary alcohols was better than those of other yeast ADH; however, the enzyme was less sensitive toward inhibitors. Kinetic studies indicated that C. guilliermondii ADH1 oxidized ethanol by an ordered bi–bi mechanism, with NAD as the first substrate fixed. Key words: Candida guilliermondii, alcohol dehydrogenase, ADH1, tetrameric.

Studies of NADP+-preferred secondary alcohol dehydrogenase from Thermoanaerobium brockii

1990 ◽  
Vol 68 (6) ◽  
pp. 907-913 ◽  
Author(s):  
Loola S. Al-Kassim ◽  
C. Stan Tsai
Keyword(s):  
Alcohol Dehydrogenase ◽  
Turnover Rate ◽  
Dissociation Constants ◽  
Secondary Alcohol ◽  
Kinetic Studies ◽  
Enzymic Activity ◽  
Secondary Alcohols ◽  
High Turnover ◽  
Subunit Molecular Weight ◽  
Secondary Alcohol Dehydrogenase

Alcohol dehydrogenase has been purified from the cell-free preparation of Thermoanaerobium brockii to homogeneity, employing combined DEAE, Sephadex, and affinity chromatographic procedures. The enzyme is tetrameric having subunit molecular weight of 40.4 × 103. The purified alcohol dehydrogenase is capable of utilizing either NAD+ or NADP+ to oxidize primary and secondary alcohols, although it prefers NADP+ as the coenzyme and secondary alcohols as substrates. Inactivation of the enzymic activity by sensitized photooxidation and carboxymethylation implicates the presence of catalytically important histidine and cysteine residues. Kinetic studies indicate that Thermoanaerobium alcohol dehydrogenase catalyzes NADP+-linked oxidations of secondary alcohols by an ordered bi-bi mechanism with NADP+ as the leading reactant. The preference of the Thermoanaerobium enzyme for NADP+ is correlated with its low dissociation constants (KA and KiA) and high turnover rate (V/Et). The corresponding kinetic parameters also contribute to the preference of this enzyme for secondary alcohols.Key words: NADP+-preferred secondary alcohol dehydrogenase.

scholarly journals Kinetics and reaction mechanism of yeast alcohol dehydrogenase with long-chain primary alcohols

1976 ◽  
Vol 157 (1) ◽  
pp. 15-22 ◽  
Author(s):  
W Schöpp ◽  
H Aurich
Keyword(s):  
Alcohol Dehydrogenase ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Random Order ◽  
Primary Alcohols ◽  
Yeast Alcohol Dehydrogenase ◽  
Kinetic Coefficients ◽  
Non Linear ◽  
High Concentrations ◽  
Long Chain

Kinetic studies of yeast alcohol dehydrogenase with NAD+ and ethanol, hexanol or decanol as substrates invariably result in non-linear Lineweaver-Burk plots if the alcohol is the variable substrate. The kinetic coefficients determined from secondary plots are consistent with an ‘equilibrium random-order‘ mechanism for extremely low alcohol concentrations and for all alcohols, the transformation of the ternary complexes being the rate-limiting step of the reaction. This mechanism also applies to long-chain substrates at high concentrations, whereas the rate of the ethanol-NAD+ reaction at high ethanol concentrations is determined by the dissociation of the enzyme-NADH complex. The dissociation constants for the enzyme-NAD+ complex and for the enzyme-alcohol complexes obtained from the kinetic quotients satisfactorily correspond to the dissociation constants obtained by use of other techniques. It is suggested that the non-linear curves may be attributed to a structural change in the enzyme itself, caused by the alcohol.

scholarly journals 4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2

1991 ◽  
Vol 274 (3) ◽  
pp. 833-842 ◽  
Author(s):  
H H Locher ◽  
T Leisinger ◽  
A M Cook
Keyword(s):  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Comamonas Testosteroni ◽  
Visible Absorption ◽  
Mononuclear Iron ◽  
Purification And Properties ◽  
A Subunit ◽  
Protein Components

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

Purification and kinetic characterization of pickerel liver alcohol dehydrogenase with dual coenzyme specificity

1993 ◽  
Vol 71 (9-10) ◽  
pp. 421-426 ◽  
Author(s):  
Loola S. Al-Kassim ◽  
C. Stan Tsai
Keyword(s):  
Alcohol Dehydrogenase ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Resonance Spectroscopy ◽  
Coenzyme Specificity ◽  
Liver Alcohol Dehydrogenase ◽  
Steady State Kinetic ◽  
Dehydrogenase Isozyme ◽  
State Kinetic

A major alcohol dehydrogenase isozyme that displays dual coenzyme specificity has been purified from pickerel liver by ion-exchange, gel filtration, and affinity chromatographic procedures. The purified enzyme is chromatographically and electrophoretically homogeneous. It is dimeric and possesses common physical properties shared by other liver alcohol dehydrogenases. Phosphorus-31 nuclear magnetic resonance spectroscopy demonstrates that NADP+ binds to two coenzyme sites of the pickerel enzyme. Steady-state kinetic studies suggest that pickerel liver alcohol dehydrogenase catalyzes NAD(P)+-linked ethanol oxidation via a random pathway. While the NADP+ reduction involves the formation of an abortive complex at high NADP+ concentrations, the NAD+ reduction at low NAD+ concentrations follows an ordered Bi-Bi mechanism with NAD+ being the leading reactant.Key words: purification, pickerel liver, alcohol dehydrogenase.

scholarly journals Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 483-496 ◽  
Author(s):  
C S Tsai
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Primary Alcohols ◽  
Reductive Methylation ◽  
Factors Affecting ◽  
Liver Alcohol Dehydrogenase ◽  
Wide Range ◽  
Lysine Residues ◽  
Michaelis Constants

Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues.

Purification and Properties of Acridone Synthase from Cell Suspension Cultures of Ruta gvaveolens L.

1994 ◽  
Vol 49 (1-2) ◽  
pp. 26-32 ◽  
Author(s):  
Alfred Baumert ◽  
Walter Maier ◽  
Detlef Gröger ◽  
Rainer Deutzmann
Keyword(s):  
Cell Suspension ◽  
Gel Filtration ◽  
Cell Suspension Cultures ◽  
Apparent Molecular Weight ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Subunit Structure ◽  
Purification And Properties ◽  
A Subunit

Acridone synthase has been purified from cell suspension cultures of Ruta graveolens using a combination of gel filtration and ion exchange chromatography. The purified enzyme has an apparent molecular weight of 69 kDa on gel filtration and a subunit structure on SDS-PAGE of 40 kDa. The apparent Km-values are 10.64 μM and 32.8 μM for N-methylanthraniloyl-CoA and malonyl-CoA, respectively. Tryptic digestion of the homogeneous acridone synthase was performed. Seven of the peptides were chosen for microsequencing. The homology of the amino acid sequences from this particular polypeptide and corresponding peptides from chalcone synthase 3 from garden pea amounted to 76%.

scholarly journals Enzyme instability and proteolysis during the purification of an alcohol dehydrogenase from Drosophila melanogaster

1977 ◽  
Vol 163 (2) ◽  
pp. 317-323 ◽  
Author(s):  
D R Thatcher
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Evolutionary Significance ◽  
Nitrobenzoic Acid ◽  
A Subunit ◽  
Terminal Amino

The alcohol dehydrogenase of the Drosophila melanogaster adhUF allele (alloenzyme with ultra-fast electrophoretic mobility) was unstable in crude or partially purified preparations. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that inactivation was porbably due to proteolytic degradation, and new method of purification of the enzyme was developed. After three steps, namely salmine sulphate precipitation, hydroxyapatite chromatography and Sephadex G-100 gel filtration, a 10-fold purified preparation was obtained. The enzyme produced was relatively stable compared with alcohol dehydrogenase purified by other methods, and was shown to be proteinase-free. The enzyme had a subunit mol.wt. of 24000 and had a single thiol residue per subunit available for titration with 5,5′-dithiobis-(2-nitrobenzoic acid). The amino acid composition and C-terminal amino acid sequence of the enzyme were determined. The substrate specificity of this alcohol dehydrogenase was also characterized. These results are discussed in relation to experiments on the evolutionary significance of thermostability at the adh locus.

scholarly journals Purification and properties of pantohenase from Pseudomonas fluorescens

1976 ◽  
Vol 157 (2) ◽  
pp. 409-413 ◽  
Author(s):  
R K Airas ◽  
E A Hietanen ◽  
V T Nurmikko
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Fluorescens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Purification And Properties

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.

scholarly journals Purification and properties of an alginate lyase from a marine bacterium

1976 ◽  
Vol 159 (3) ◽  
pp. 707-713 ◽  
Author(s):  
I W Davidson ◽  
I W Sutherland ◽  
C J Lawson
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alginate Lyase ◽  
Kinetic Studies ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Bacterial Cells ◽  
Purification And Properties ◽  
Primary Composition

An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples.

scholarly journals Biochemical properties of alcohol dehydrogenase from Drosophila lebanonensis

1986 ◽  
Vol 235 (2) ◽  
pp. 481-490 ◽  
Author(s):  
J O Winberg ◽  
R Hovik ◽  
J S McKinley-McKee ◽  
E Juan ◽  
R Gonzalez-Duarte
Keyword(s):  
Alcohol Dehydrogenase ◽  
Active Site ◽  
Active Sites ◽  
Electrophoretic Pattern ◽  
Maximum Velocity ◽  
Cathode Side ◽  
Secondary Alcohols ◽  
Starch Gel Electrophoresis ◽  
Primary Alcohols ◽  
Rate Limiting

Purified Drosophila lebanonensis alcohol dehydrogenase (Adh) revealed one enzymically active zone in starch gel electrophoresis at pH 8.5. This zone was located on the cathode side of the origin. Incubation of D. lebanonensis Adh with NAD+ and acetone altered the electrophoretic pattern to more anodal migrating zones. D. lebanonensis Adh has an Mr of 56,000, a subunit of Mr of 28 000 and is a dimer with two active sites per enzyme molecule. This agrees with a polypeptide chain of 247 residues. Metal analysis by plasma emission spectroscopy indicated that this insect alcohol dehydrogenase is not a metalloenzyme. In studies of the substrate specificity and stereospecificity, D. lebanonensis Adh was more active with secondary than with primary alcohols. Both alkyl groups in the secondary alcohols interacted hydrophobically with the alcohol binding region of the active site. The catalytic centre activity for propan-2-ol was 7.4 s-1 and the maximum velocity of most secondary alcohols was approximately the same and indicative of rate-limiting enzyme-coenzyme dissociation. For primary alcohols the maximum velocity varied and was much lower than for secondary alcohols. The catalytic centre activity for ethanol was 2.4 s-1. With [2H6]ethanol a primary kinetic 2H isotope effect of 2.8 indicated that the interconversion of the ternary complexes was rate-limiting. Pyrazole was an ethanol-competitive inhibitor of the enzyme. The difference spectra of the enzyme-NAD+-pyrazole complex gave an absorption peak at 305 nm with epsilon 305 14.5 × 10(3) M-1 × cm-1. Concentrations and amounts of active enzyme can thus be determined. A kinetic rate assay to determine the concentration of enzyme active sites is also presented. This has been developed from active site concentrations established by titration at 305 nm of the enzyme and pyrazole with NAD+. In contrast with the amino acid composition, which indicated that D. lebanonensis Adh and the D. melanogaster alleloenzymes were not closely related, the enzymological studies showed that their active sites were similar although differing markedly from those of zinc alcohol dehydrogenases.

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