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During growth, PCE was dechlorinated and the isotope ratio of PCE increased from. units for the isotope measurement is much smaller than the symbol.PCE was reduced via TCE to primarily cisDCE.The highest fractionation occurred using the puried reductive dehalogenase, suggesting that cell components might affect the fractionation and cause a decrease of the overall C.Figure E and F shows the reductive dechlorination of PCE by reduced cyanocobalamin.TCE was the major product, and minor quantities of cisDCE were observed.Cobalamins are important cofactors in <a href="http://www.targetmol.com/compound/Dyclonine-hydrochloride"></a> microorganisms capable of dehalorespiration. The extent of the carbon isotope effect, which points to a primary kinetic isotope effect, suggests that the ratelimiting step in the dehalogenation reaction is probably controlled by cleavage of a bond.The kinetic isotope effect of hydrogen was much lower, suggesting a secondary isotope effect, which indicates that the protonation was not rate limiting during the overall reaction and may occur subsequent to the dehalogenation.Identication of chloroethenylcobalamins as intermediates provides evidence for the formation of a covalent cobaltcarbon bond during dehalogenation indicates that a chlorine has already been released, suggesting an irreversible reaction step.Therefore, the subsequent cleavage of the carboncobalt bond is probably not a ratelimiting step in the dehalogenation reaction.Although the number of intermediate steps in unidirectional biochemical reactions may be unknown, the kinetic isotope fractionation should be caused by the rst irreversible reaction step in the reaction sequence associated with the conformational change of a chemical bond.In a twostep reaction, the partitioning factor using equation, below, assuming that the preceding reaction steps are rate limiting to some extent but not associated with a signicant isotope effect.This assumption is reasonable because preceding reaction steps such as uptake and the binding of a substrate are usually not associated with a change of bond conformation.With the assumption that the isotope fractionation is caused by the dehalogenation reaction at the cobalamin, signicant primary isotope effects may be masked by physiological factors, lowering the extent of the overall observed isotope fractionation.Hence, it is extremely difcult to analyze the transition   state and mechanism of the biochemical reaction just by using isotope fractionation factors obtained from culture experiments.It should, however, be noted that the cobalamin cofactor of the PCE dehalogenase of this organism is a novel and unusual type of corrinoid. The molecular structure of the enzyme is not known, but probably binding of the reactant to the enzyme complex and coordination of the reactant in the reactive center affects the rate of the overall reaction.Sorption did not affect the isotope composition signicantly; however, sorption may affect the reaction kinetic of dehalogenation.Alternatively, the coordination of the reactant in the enzyme complex can change activation energy and thus the reaction kinetic and may thereby affect the isotope fractionation.PCES, with reductions in isotope fractionation of and, respectively, comparing growing cells to crude extracts.Therefore, the membrane may act as a barrier separating PCE pools inside and outside of the cell.If there is a transport limitation over the membrane, it can be expected that the isotope discrimination will be decreased due to a nearly complete consumption of the PCE available inside the cell.

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