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

Dickinson, F M; Dickenson, C J

doi:10.1042/bj1710629

Cited by 24 publications

(36 citation statements)

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“…It is not unusual that the product of the hydride-transfer step should exhibit little fluorescence. This has been seen also with liver aldehyde dehydrogenase , glutamate dehydrogenase (di Franco & Iwatsubo, 1972), lactate dehydrogenase (Whitaker et al, 1974) and yeast alcohol dehydrogenase (Dickinson & Dickenson, 1978).…”

Section: Resultsmentioning

confidence: 91%

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae

Dickinson

Haywood

1987

Biochemical Journal

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The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process. Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4 %) in the complete absence of K+. The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH. Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex. The variation of activity of the enzyme with pH was

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Section: Resultsmentioning

confidence: 91%

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae

Dickinson

Haywood

1987

Biochemical Journal

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“…The equilibrium in solution favors the alcohol-NAD þ side of the reaction (at catalytically relevant pH) (7), but the solution equilibrium is not directly relevant to the rate-limiting hydride transfer step under study. Measurements of internal (on the enzyme) equilibrium gave a value of K eq ¼ 0.15 in favor of the alcohol-NAD þ side, predicting a late TRS (9), but the internal equilibrium is close to unity and very likely a combination of at least two steps: the deprotonation of the alcohol and the hydride transfer that follows (46). Indeed, QM/MM calculations for this reaction found that the hydride transfer step is exothermic, despite the fact that the overall reaction was endothermic (24,27).…”

Section: Resultsmentioning

confidence: 99%

“…Intriguingly, decades of experiments on the reaction catalyzed by yADH have returned what appear to be contradictory results, some suggesting an early TS, whereas others point to a late TS (7)(8)(9)(10). In pioneering studies, Klinman measured linear free energy relationships (LFERs) by using benzyl substrates in both forward and reverse directions and found evidence to support an alcohol-like TS (7,8), despite the fact that Hammond's postulate predicts a late TS for this slightly endothermic reaction (internal K eq ¼ 0.15, i.e., ∼1 kcal∕mol) (9).…”

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confidence: 99%

“…In pioneering studies, Klinman measured linear free energy relationships (LFERs) by using benzyl substrates in both forward and reverse directions and found evidence to support an alcohol-like TS (7,8), despite the fact that Hammond's postulate predicts a late TS for this slightly endothermic reaction (internal K eq ¼ 0.15, i.e., ∼1 kcal∕mol) (9). Klinman also examined 2°ki-netic isotope effects (KIEs) only to implicate an aldehyde-like TS (8).…”

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confidence: 99%

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Elusive transition state of alcohol dehydrogenase unveiled

Roston

Kohen

2010

Proc. Natl. Acad. Sci. U.S.A.

154

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For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (>1) 2°kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (<1), this KIE eludes the traditional interpretation of 2°KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate.hydrogen tunneling | enzyme kinetic | secondary isotope effect | Swain-Schaad E nzymes enhance the rates of chemical reactions by many orders of magnitude, and extensive studies have uncovered many aspects of how they do so. The prevailing theory that enzymes stabilize the transition state (TS) relative to the reactants explains many phenomena, and a great deal of contemporary research focuses on how enzymes stabilize the TS. An unfortunate hindrance to developing an understanding of how enzymes stabilize the TS lies in the enigmatic nature of the TS. Many enzymatic hydrogen (proton, hydrogen, or hydride) transfers, for example, occur by quantum mechanical tunneling, where a particle passes through an energy barrier because of its wave properties (1-6).Yeast alcohol dehydrogenase (yADH) serves as an excellent model system for enzyme-catalyzed H transfer because unlike many other enzymes, the chemical step, oxidation of a primary alcohol to an aldehyde by NAD þ , is rate-limiting with aromatic substrates (7). Furthermore, the thermodynamics of the reaction allow for examination of both the forward (alcohol to aldehyde) and reverse (aldehyde to alcohol) reactions under similar conditions (8). This type of nicotinamide-dependent redox reaction appears ubiquitously in biology, and a detailed picture of the TS of such reactions may facilitate many medical and industrial applications.Intriguingly, decades of experiments on the reaction catalyzed by yADH have returned what appear to be contradictory results, some suggesting an early TS, whereas others point to a late TS (7-10). In pioneering studies, Klinman measured linear fr...

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“…With lower unbranched aliphatic alcohols, the order of addition of substrates is steady-state random on the alcohol side and strictly ordered on the aldehyde side of the catalytic cycle [24,25]. With slow,`nonsticky' carbonyl substrates, such as acetone [8], NDMA, a benzaldehyde analog [10] and DACA [14], the order of addition of substrates on the aldehyde side is also steady-state random.…”

Section: R E S U L T S Kinetic Mechanismmentioning

confidence: 99%

Comparison of the chemical mechanisms of action of yeast and equine liver alcohol dehydrogenase

Leskovac

Trivić

Anderson

1999

European Journal of Biochemistry

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The pH‐dependence of the steady‐state kinetic parameters and the ligand‐binding parameters for competitive dead‐end inhibitors for the yeast alcohol dehydrogenase (, constitutive, cytoplasmic) reaction was studied in the pH range 6–10. These studies were designed in order to assign the appropriate pKa values to all dissociation forms of enzyme in the chemical mechanism of action for the yeast enzyme, previously proposed by Cook and Cleland [P. F. Cook & W. W. Cleland (1981) Biochemistry20, 1796–1816]. In addition, the chemical mechanism of action for the yeast enzyme, proposed in this work, was compared with a similar mechanism of action for the horse liver enzyme, proposed by Cook and Cleland. Substantial differences were found, especially in the binding of coenzymes and in the structure of enzyme–coenzyme complexes.

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

Abstract: Stopped-flow studies of oxidation of butan-l-ol and propan-2-ol by NAD+

Cited by 24 publications

References 20 publications

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae

Elusive transition state of alcohol dehydrogenase unveiled

Comparison of the chemical mechanisms of action of yeast and equine liver alcohol dehydrogenase

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