Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70°C

Guagliardi, Annamaria; Martino, Matilde; Iaccarino, Ingram; Rosa, Mario De; Rossi, Mosè; Bartolucci, Simonetta

doi:10.1016/1357-2725(95)00138-7

Cited by 55 publications

(21 citation statements)

References 21 publications

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“…Hence, substrate KIEs (KIE= k cat H / k cat D ) can be determined by measuring the steady‐state rate constant ( k cat ) using protiated and deuterated substrates. In agreement with previous work,6a, 7c the resulting substrate KIE is highly temperature‐dependent, increasing sharply at lower temperatures but remaining constant above 40 °C (Figure 2 A and Figures S1, S2 in the Supporting Information). The natural substrates of BsADH are generally small molecules, which serve as hydride acceptors under anaerobic conditions 9.…”

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confidence: 92%

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

et al. 2018

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The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate‐dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.

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supporting

confidence: 92%

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

et al. 2018

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“…The alcohol dehydrogenase (ADH) family exists widely in the genomes of bacteria, insects, plants, and vertebrates (Guagliardi et al, 1996;Fischer and Maniatis, 1985;Martinez et al, 1996;Barth and Kunkel, 1979;Canestro et al, 2000;Reimers et al, 2004). All ADH classes form dimers and catalyze oxidization of various kinds and concentrations of alcohols using NAD + /NADH as coenzyme (Eklund et al, 1976a,b;Höög et al, 2001).…”

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confidence: 98%

Conservative evolution in duplicated genes of the primate Class I ADH cluster

Oota

Dunn

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et al. 2007

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“…This point should be emphasized for enzymatic reactions: do the rates measured actually represent a meaningful consistent rate constant over the whole temperature range? For example, in an early report of convex Arrhenius plot for the thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ADH-hT), all measurements were conducted with the same substrate concentration (that was saturating the enzyme at room temperature), and V max was presented (47). A later report (22) found that K M increases with temperature so at high temperature [S] was not sufficiently larger than K M , and the measured rate was found to be slower than V max , presenting an apparently convex Arrhenius plot.…”

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confidence: 99%

Convex Arrhenius plots and their interpretation

Truhlar

Kohen

2001

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

151

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This paper draws attention to selected experiments on enzymecatalyzed reactions that show convex Arrhenius plots, which are very rare, and points out that Tolman's interpretation of the activation energy places a fundamental model-independent constraint on any detailed explanation of these reactions. The analysis presented here shows that in such systems, the rate coefficient as a function of energy is not just increasing more slowly than expected, it is actually decreasing. This interpretation of the data provides a constraint on proposed microscopic models, i.e., it requires that any successful model of a reaction with a convex Arrhenius plot should be consistent with the microcanonical rate coefficient being a decreasing function of energy. The implications and limitations of this analysis to interpreting enzyme mechanisms are discussed. This model-independent conclusion has broad applicability to all fields of kinetics, and we also draw attention to an analogy with diffusion in metastable fluids and glasses. In recent years, an increasing number of studies have measured the temperature dependence of enzyme reactions for both thermostable and mesostable enzymes (enzymes from organisms that grow optimally at high temperature and at normal body temperature, respectively). The temperature dependence is usually exhibited in an Arrhenius plot, which is the natural logarithm of the rate coefficient vs. reciprocal of the absolute temperature ( Fig. 1). Some of the methodologies for measuring the temperature dependences of reaction rates have been applied to measure the temperature dependences of the kinetic isotope effects (KIEs) of the catalyzed reactions as well, and such experiments provide experimental data very relevant to characterizing the dynamical bottlenecks of the reactions (1-6). A large number of theoretical models have been advanced for the interpretation of this kind of data (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).Experiments on reactions catalyzed by a variety of thermostable and mesostable dehydrogenase and oxidase enzymes have shown that in some cases the Arrhenius plot for the chemical step is convex (20)(21)(22)(23). In the present paper, we comment on the significance of this observation, and we provide an interpretation of this finding that places it in the broader perspective of nonbiological chemical as well as biochemical kinetics. This paper points out that the convex Arrhenius behavior places a severe constraint on the microcanonical unimolecular rate coefficient for the chemical step. In particular, we can go beyond the obvious interpretation that a convex Arrhenius plot can be interpreted as a lower than expected rate at high temperatures, and we can say, under certain conditions, that the microcanonical unimolecular rate coefficient for the chemical step must actually decrease with increasing energy.In general, it is desirable to separate the interpretation of experimental data into two steps. First, we infer general consequences that rest on solid foundations such as thermody...

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Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70°C

Cited by 55 publications

References 21 publications

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

Conservative evolution in duplicated genes of the primate Class I ADH cluster

Convex Arrhenius plots and their interpretation

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