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 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 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 Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases

1978 ◽  
Vol 171 (3) ◽  
pp. 613-627 ◽  
Author(s):  
C J Dickenson ◽  
F M Dickinson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Dissociation Constant ◽  
Maximum Rate ◽  
Alternative Pathway ◽  
Product Inhibition ◽  
Experimental Conditions ◽  
Alcohol Dehydrogenases ◽  
Yeast Alcohol Dehydrogenase ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.

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.

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 A study of the ionic properties of the essential histidine residue of yeast alcohol dehydrogenase in complexes of the enzyme with its coenzymes and substrates

1977 ◽  
Vol 161 (1) ◽  
pp. 73-82 ◽  
Author(s):  
C J Dickenson ◽  
F M Dickinson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Initial Rate ◽  
Dissociation Constants ◽  
Ternary Complexes ◽  
Histidine Residue ◽  
Mechanism Of Formation ◽  
Binary And Ternary Complexes ◽  
Yeast Alcohol Dehydrogenase ◽  
Ph Range

1. Initial-rate studies of the reduction of acetaldehyde by NADH, catalysed by yeast alcohol dehydrogenase, were performed at pH 4.9 and 9.9, in various buffers, at 25 degrees C. The results are discussed in terms of the mechanism previously proposed for the pH range 5.9-8.9 [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. 2. Acetaldehyde forms a u.v.-absorbing complex with glycine. This was shown not to affect the results of kinetic experiments under the conditions used in this and earlier work. 3. The variation with pH of the dissociation constant for the enzyme-NADH complex, calculated from the initial-rate data, indicates that the enzyme possesses a group with pK7.1 in the free enzyme and pK8.7 in the complex. 4. The pH-dependences of the second-order rate constants for inactivation of the enzyme by diethyl pyrocarbonate were determined for the free enzymes (pK7.1), the enzyme-NAD+ complex (pK approx. 7.1) and the enzyme-NADH complex (pK approx. 8.4). The essential histidine residue may therefore be the group involved in formation and dissociation of the enzyme-NADH complex. 5. Estimates of the rate constant for reaction of acetaldehyde with the enzyme-NADH complex indicate that acetaldehyde may combine only when the essential histidine residue is protonated. The dissociation constants for butan-1-ol and propan-2-ol, calculated on the basis of earlier kinetic data, are, however, independent of pH. 6. The results obtained are discussed in relation to the role of the essential histidine residue in the mechanism of formation of binary and ternary complexes of the enzyme with its coenzymes and substrates.

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