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 Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

1978 ◽  
Vol 171 (3) ◽  
pp. 629-637 ◽  
Author(s):  
F. Mark Dickinson ◽  
Christopher J. Dickenson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Catalytic Mechanism ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Product Inhibition ◽  
Stopped Flow ◽  
Phenol Red ◽  
Yeast Alcohol Dehydrogenase ◽  
Fluorescence Measurements ◽  
High Concentrations

Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD+ in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pKa approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185–4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD+ release from the enzyme–NAD+–ethanol product complex. This finding permits calculation of K3, the dissociation constant for ethanol from the enzyme–NAD+–ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613–627). The calculations show that K3 varies very little with pH in the range 5.95–8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme–NADH–acetaldehyde]/ [enzyme–NAD+–ethanol] at equilibrium. The values obtained were in the range 0.11±0.04, and this value together with estimates of K3 was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.

scholarly journals Kinetic studies of glutamate dehydrogenase. The reductive amination of 2-oxoglutarate

1970 ◽  
Vol 118 (3) ◽  
pp. 409-419 ◽  
Author(s):  
P. C. Engel ◽  
K. Dalziel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Initial Rate ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Random Order ◽  
Order Type ◽  
Reverse Reaction ◽  
Enzyme Subunits ◽  
High Concentrations

1. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH7.0 and pH 8.0. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Lineweaver–Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. 2. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. On the basis of this mechanism, the dissociation constants of all the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. 3. The results are discussed in relation to those of earlier workers who concluded that the mechanism is of the compulsory-order type.

Relative reactivities of primary alcohols as substrates of liver alcohol dehydrogenase

1968 ◽  
Vol 46 (4) ◽  
pp. 381-385 ◽  
Author(s):  
C. S. Tsai
Keyword(s):  
Alcohol Dehydrogenase ◽  
Rate Equation ◽  
Electronic Effect ◽  
Graphical Analysis ◽  
Primary Alcohols ◽  
Structural Requirement ◽  
Liver Alcohol Dehydrogenase ◽  
Polar Groups ◽  
Kinetic Coefficients ◽  
Kinetics Of

To obtain information concerning the general structural requirement of alcohols as substrates of liver alcohol dehydrogenase, the kinetics of the enzymic oxidation of primary alcohols were studied. All the active substrates possess hydrophobic side groups. Introduction of polar groups renders alcohols inactive or inhibitory. To correlate reactivities of primary alcohols as substrates with the nature of their side groups, their relative reactivities are expressed in terms of kinetic coefficients which were solved from the rate equation by successive graphical analysis. Based on kinetic results, the relative reactivities of primary alcohols as substrates of liver alcohol dehydrogenase are discussed in relation to the hydrophobic interaction and the electronic effect of their side groups.

scholarly journals The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides

1972 ◽  
Vol 126 (4) ◽  
pp. 975-984 ◽  
Author(s):  
K. Dalziel ◽  
R. R. Egan
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Kinetic Studies ◽  
Binding Studies ◽  
Purine Nucleotides ◽  
Active Centre ◽  
Sodium Phosphate Buffer ◽  
Low Concentrations ◽  
High Concentrations

1. The binding of NAD+ and NADP+ to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD+ the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD+ and NADP+, in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD+ or NADP+, the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.

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.

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.

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.

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