Untersuchungen zur Reaktion der Alkoholdehydrogenase mit Tritium -markierten Substraten

1966 ◽  
Vol 21 (6) ◽  
pp. 547-551 ◽  
Author(s):  
Dieter Palm
Keyword(s):  
Alcohol Dehydrogenase ◽  
Isotope Effect ◽  
Isotope Fractionation ◽  
Kinetic Isotope Effect ◽  
Retention Volume ◽  
Deae Cellulose ◽  
Yeast Alcohol Dehydrogenase ◽  
Kinetic Isotope ◽  
Direct Indication ◽  
Rate Controlling Step

The temperature dependence of the kinetic isotope effect of NADH-T in the acetaldehyde reduction by yeast alcohol dehydrogenase showed a discontinuity which can be explained by a change of the rate controlling step. The magnitude of the isotope effect is largely dependent on the nature of the unlabelled aldehyde and increases in the order acetaldehyde, propionaldehyde, butyraldehyde. This is a direct indication that the second substrate influences the nature of the H-transfer from NADH. During substrate binding the aldehyde causes an effect on the transferable H of NADH. This effect is less pronounced for the less effective substrates propionaldehyde and butyraldehyde. Comparing the homologue aldehydes the small size of the isotope effect gives an indication that acetaldehyde is the natural substrate of yeast alcohol dehydrogenase.The purification of NADH-T on DEAE-Cellulose is connected with isotope fractionation which amounts to 0.4 — 1.1% of retention volume.

Untersuchungen zur Reaktion der Alkoholdehydrogenase mit Tritium-markierten Substraten

1966 ◽  
Vol 21 (6) ◽  
pp. 540-546 ◽  
Author(s):  
Dieter Palm
Keyword(s):  
Alcohol Dehydrogenase ◽  
Temperature Dependence ◽  
Isotope Effect ◽  
Substrate Binding ◽  
Isotope Effects ◽  
Direct Interaction ◽  
Enzyme Complex ◽  
Temperature Range ◽  
Yeast Alcohol Dehydrogenase ◽  
Rate Controlling Step

Unexpectedly, the isotope effect of ethanol-1-Τ as a substrate of yeast alcohol dehydrogenase, increases with rising temperature from kH/kT = 3.2 at 5 —15°C to 3.8—4.7 at 20 —35 °C. This suggests a change of the rate controlling step as proposed by MÜLLER-HILL and WALLENFELS, who investigated the temperature dependence of the activation energies in this temperature range. A comparison of the affinities of propanol and butanol with the isotope effects of the corresponding tritium labelled compounds (propanol-1-Τ 6.7 at 25 °C, butanol-1-Τ 6.8 at 25 °C) supports the proposal, that during substrate binding, there must be a direct interaction between the enzyme complex and hydrogen which is removed in the reaction. These influences are less pronounced for the ethanol homologues which are bound less tightly to the enzyme. Therefore the H transfering step proper gives a greater contribution to the overall experimental isotope effect.

Fundamental studies on kinetic isotope effect (KIE) of hydrogen isotope fractionation in natural gas systems

2011 ◽  
Vol 75 (10) ◽  
pp. 2696-2707 ◽  
Author(s):  
Yunyan Ni ◽  
Qisheng Ma ◽  
Geoffrey S. Ellis ◽  
Jinxing Dai ◽  
Barry Katz ◽  
...  
Keyword(s):  
Natural Gas ◽  
Isotope Effect ◽  
Hydrogen Isotope ◽  
Isotope Fractionation ◽  
Kinetic Isotope Effect ◽  
Kinetic Isotope

scholarly journals Evidence of Substantial Carbon Isotope Fractionation among Substrate, Inorganic Carbon, and Biomass during Aerobic Mineralization of 1,2-Dichloroethane byXanthobacter autotrophicus

2000 ◽  
Vol 66 (11) ◽  
pp. 4870-4876 ◽  
Author(s):  
D. Hunkeler ◽  
R. Aravena
Keyword(s):  
Carbon Isotope ◽  
Isotope Effect ◽  
Isotope Fractionation ◽  
Kinetic Isotope Effect ◽  
Inorganic Carbon ◽  
Mechanistic Model ◽  
Fractionation Factor ◽  
Carbon Isotope Fractionation ◽  
Kinetic Isotope ◽  
Factor Α

ABSTRACT Carbon isotope fractionation during aerobic mineralization of 1,2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicusGJ10 was investigated. A strong enrichment of 13C in residual 1,2-DCA was observed, with a mean fractionation factor α ± standard deviation of 0.968 ± 0.0013 to 0.973 ± 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor α to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of 13C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an SN2 nucleophilic substitution. SN2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.

scholarly journals Anaerobic methane oxidation by nitrate: kinetic isotope effect

2018 ◽  
Vol 10 (1) ◽  
Author(s):  
Vasiliy A. Vavilin
Keyword(s):  
Dynamic Model ◽  
Carbon Isotope ◽  
Carbon Isotopes ◽  
Isotope Effect ◽  
Isotope Fractionation ◽  
Kinetic Isotope Effect ◽  
Stable Carbon Isotopes ◽  
Stable Carbon ◽  
Fractionation Factor ◽  
Kinetic Isotope

The ratio of stable carbon isotopes (13C/12C) in different environments serves as a significant limitation in estimating the global balance of methane [Hornibrook et al., 2000]. In this case, the value of 13C/12C largely depends on the kinetic isotope effect associated with the metabolism of microorganisms that produce and consume CH4. The article suggests a dynamic model of the processes of methane formation and its anaerobic oxidation with nitrate by methanotrophic denitrifying microorganisms (DAOM), which allowed estimating the fractionation factor of stable carbon isotopes. In the experiment with peat from the minerotrophic bog [Smemo, Yavitt, 2007], the dynamics of the amount of methane and was measured. The dynamic model showed that the introduction of nitrate leads to a slow decrease in the partial pressure of methane. Since methane in the DAOM process is a substrate, methane is enriched with heavier carbon 13C in the system under study. This leads to an increase in the value . The carbon isotope fractionation factor during methane oxidation with nitrate was equal to 1.018 and comparable with the fraction of carbon isotope fractionation in the process of acetoclastic methanogenesis (1.01). Model calculations have shown that during incubation the apparent fractionation factor of carbon isotopes with the simultaneous formation of methane and DAOM slowly decreases. The ratio of 13C/12C isotopes in dissolved and gaseous methane practically does not differ. The model showed that an increase in the initial concentration of nitrate increases the rate of DAOM, which leads to a decrease in the concentration of dissolved methane. In this case, the value of 13C/12C increases. In field studies, Shi et al. (2017) showed that the presence of DAOM in peat bogs in which fertilizers penetrate can be controlled by the amount of nitrate used and the depth of penetration into the anoxic layer. Two MATLAB files describing DAOM are attached to the article.

Quantum electrocatalysts: theoretical picture, electrochemical kinetic isotope effect analysis, and conjecture to understand microscopic mechanisms

2020 ◽  
Vol 22 (20) ◽  
pp. 11219-11243 ◽  
Author(s):  
Ken Sakaushi
Keyword(s):  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Effect Analysis ◽  
Kinetic Isotope

The fundamental aspects of quantum electrocatalysts are discussed together with the newly developed electrochemical kinetic isotope effect (EC-KIE) approach.

scholarly journals l-mandelate dehydrogenase from Rhodotorula graminis: comparisons with the l-lactate dehydrogenase (flavocytochrome b2) from Saccharomyces cerevisiae

1993 ◽  
Vol 290 (1) ◽  
pp. 103-107 ◽  
Author(s):  
O Smékal ◽  
M Yasin ◽  
C A Fewson ◽  
G A Reid ◽  
S K Chapman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactate Dehydrogenase ◽  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Dimensional Structure ◽  
Striking Difference ◽  
Kinetic Properties ◽  
Proton Abstraction ◽  
Rate Determining Step ◽  
Kinetic Isotope

L-Lactate dehydrogenase (L-LDH) from Saccharomyces cerevisiae and L-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis are both flavocytochromes b2. The kinetic properties of these enzymes have been compared using steady-state kinetic methods. The most striking difference between the two enzymes is found by comparing their substrate specificities. L-LDH and L-MDH have mutually exclusive primary substrates, i.e. the substrate for one enzyme is a potent competitive inhibitor for the other. Molecular-modelling studies on the known three-dimensional structure of S. cerevisiae L-LDH suggest that this enzyme is unable to catalyse the oxidation of L-mandelate because productive binding is impeded by steric interference, particularly between the side chain of Leu-230 and the phenyl ring of mandelate. Another major difference between L-LDH and L-MDH lies in the rate-determining step. For S. cerevisiae L-LDH, the major rate-determining step is proton abstraction at C-2 of lactate, as previously shown by the 2H kinetic-isotope effect. However, in R. graminis L-MDH the kinetic-isotope effect seen with DL-[2-2H]mandelate is only 1.1 +/- 0.1, clearly showing that proton abstraction at C-2 of mandelate is not rate-limiting. The fact that the rate-determining step is different indicates that the transition states in each of these enzymes must also be different.

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