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.

Observations on the histochemical distribution of alcohol dehydrogenase

1965 ◽  
Vol s3-106 (76) ◽  
pp. 289-297
Author(s):  
M. M. FERGUSON
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohols ◽  
Primary Alcohols ◽  
Straight Chain ◽  
Tertiary Alcohols ◽  
Polyhydric Alcohols ◽  
Water Side ◽  
Monohydric Alcohols ◽  
Branched Chain ◽  
Ethereal Oxygen

Alcohol dehydrogenase capable of utilizing primary alcohols (straight and branched chain, unsaturated and cellosolves), secondary alcohols, polyhydric alcohols but not tertiary alcohols is described in the rat brain, lung, heart, liver, kidney, gut, spleen, pancreas, uterus, and seminal vesicle. With the straight chain primary alcohols the extent to which the alcohols were used increased relative to increasing chain length until the optimum length of six to seven carbon atoms had been reached, whereafter activity decreased, probably due to the insolubility of the higher members of the series in water. Side groups on the chain, double bonds and ethereal oxygen atoms, all had the effect of hindering the ease of dehydrogenation of the alcohol. Secondary alcohols were suitable as substrates; however, tertiary alcohols did not seem to be satisfactory for the demonstration of alcohol dehydrogenase. Polyhydric alcohols were suitable substrates although they were not as well used as were the monohydric alcohols. Furfuryl alcohol dehydrogenase was found in all tissues and is apparently a separate enzyme.

scholarly journals Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni

1986 ◽  
Vol 234 (3) ◽  
pp. 611-615 ◽  
Author(s):  
B W Groen ◽  
M A G van Kleef ◽  
J A Duine
Keyword(s):  
Absorption Spectrum ◽  
Cytochrome C ◽  
Alcohol Dehydrogenase ◽  
Secondary Alcohols ◽  
Primary Alcohols ◽  
Optimal Activity ◽  
Alcohol Dehydrogenase Activity ◽  
Artificial Electron Acceptor ◽  
The Common ◽  
Pseudomonas Testosteroni

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name ‘quinohaemoprotein alcohol dehydrogenase’. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.

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.

Catalytic Resonance Theory: Parallel Reaction Pathway Control

2019 ◽  
Author(s):  
M. Alexander Ardagh ◽  
Manish Shetty ◽  
Anatoliy Kuznetsov ◽  
Qi Zhang ◽  
Phillip Christopher ◽  
...  
Keyword(s):  
Chemical Reactions ◽  
Active Site ◽  
Active Sites ◽  
Reaction Pathway ◽  
Control Strategies ◽  
Oscillation Amplitude ◽  
Catalytic Performance ◽  
Parallel Reaction ◽  
Resonance Theory ◽  
Static Conditions

Catalytic enhancement of chemical reactions via heterogeneous materials occurs through stabilization of transition states at designed active sites, but dramatically greater rate acceleration on that same active site is achieved when the surface intermediates oscillate in binding energy. The applied oscillation amplitude and frequency can accelerate reactions orders of magnitude above the catalytic rates of static systems, provided the active site dynamics are tuned to the natural frequencies of the surface chemistry. In this work, differences in the characteristics of parallel reactions are exploited via selective application of active site dynamics (0 < ΔU < 1.0 eV amplitude, 10<sup>-6</sup> < f < 10<sup>4</sup> Hz frequency) to control the extent of competing reactions occurring on the shared catalytic surface. Simulation of multiple parallel reaction systems with broad range of variation in chemical parameters revealed that parallel chemistries are highly tunable in selectivity between either pure product, even when specific products are not selectively produced under static conditions. Two mechanisms leading to dynamic selectivity control were identified: (i) surface thermodynamic control of one product species under strong binding conditions, or (ii) catalytic resonance of the kinetics of one reaction over the other. These dynamic parallel pathway control strategies applied to a host of chemical conditions indicate significant potential for improving the catalytic performance of many important industrial chemical reactions beyond their existing static performance.

Nonlinear Vibrations in a 2-Dimensional Protein Cluster Model with Linear Bonds

2000 ◽  
Vol 214 (1) ◽  
Author(s):  
E.G. Shidlovskaya ◽  
L. Schimansky-Geier ◽  
Yu.M. Romanovsky
Keyword(s):  
Active Site ◽  
Internal Resonance ◽  
Cluster Model ◽  
Active Sites ◽  
Nonlinear Vibrations ◽  
Nonlinear Phenomena ◽  
Two Dimensional ◽  
Dimensional Model ◽  
Two Dimensional Model

A two dimensional model for the substrate inside a pocket of an active site of an enzyme is presented and investigated as a vibrational system. The parameters of the system are evaluated for α-chymotrypsin. In the case of internal resonance it is analytically and numerically shown that the energy concentrated on a certain degree of freedom might be several times larger than in the non-resonant case. Additionally, the system is driven by harmonic excitations and again energy due to nonlinear phenomena is redistributed inhomogeneously. These results may be of importance for the determination of the rates of catalytic events of substrates bound in pockets of active sites.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

Sign in / Sign up

Export Citation Format

Share Document