Enzyme reactions proton exchange rates

Mandelate racemase, another pertinent example, catalyzes the kinetically and thermodynamically unfavorable a-carbon proton abstraction. Bearne and Wolfenden measured deuterium incorporation rates into the a-posi-tion of mandelate and the rate of (i )-mandelate racemi-zation upon incubation at elevated temperatures. From an Arrhenius plot, they obtained a for racemization and deuterium exchange rate was estimated to be around 35 kcal/mol at 25°C under neutral conditions. The magnitude of the latter indicated mandelate racemase achieves the remarkable rate enhancement of 1.7 X 10, and a level of transition state affinity (K x = 2 X 10 M). These investigators also estimated the effective concentrations of the catalytic side chains in the native protein for Lys-166, the effective concentration was 622 M for His-297, they obtained a value 3 X 10 M and for Glu-317, the value was 3 X 10 M. The authors state that their observations are consistent with the idea that general acid-general base catalysis is efficient mode of catalysis when enzyme s structure is optimally complementary with their substrates in the transition-state. See Reference Reaction Catalytic Enhancement... 

An isotope effect technique applied in enzyme-catalyzed isotope exchange experiments measuring reaction rate as a function of the [D20]/[H20] ratio. Such studies often provide information concerning the effect of the environment of the transition state on exchangeable protons. 

As discussed, AdoMet is a high energy compound, and therefore, AdoMet-dependent methylation reactions are known to be irreversible. However, enzymatic catalysis is often required to enhance the rate of the reaction. Enzymes employ a variety of ways to enhance the nucleophilicity on the attacking atom in a substrate. Often, the reaction results in a proton exchange for the methyl group. The proton can be removed before, in concert with, or after the methyl transfer this step usually requires the presence of a general base in the active site. 

No examples of the use of deuterium and tritium NMR in xenobiotic metabolism were found. Their use in biosynthetic studies has been reviewed by Garson and Staunton (31). Sensitivity problems exist with deuterium, but should not be a problem with tritium since it is the most sensitive nucleus available (1.21 x proton) and because of negligible tritium backgrounds. Tritium NMR may be useful in the studies of xenoblotic-enzyme interactions as shown by Scott et al. (32). Hazards due to the use of radioactivity should be minimal because 1 mCi of activity should provide sufficient material for many experiments. However, isotope effects may be a problem if the metabolic reaction directly involves the tritium (or deuterium) atom because Isotopes of hydrogen can greatly affect enzymic reaction rates. Also, lability may be a problem as Bakke and Feil have found with CD3SO compounds, where exchange was too rapid to permit metabolism studies (W). 

H/D exchange follows a pseudo-first order reaction. It must be noticed that the H/ D exchange rate constant at the C2-atom of enzyme-bound ThDP represents the lower limit of the C2-H deprotonation, because this observed value refiects not only the deprotonation, but also the exchange rate constant of the base responsible for the deprotonation with solvent protons. The observed rate constant of the H/D-exchange kobs is composed of the rate constant of deuteration ko of the car-banion intermediate, and that of its reprotonation kn according to the equation kobs = y (fen + ko) (Tittmann, 2000). The fractionation factor rf) of this reaction is... 

These enzymes catalyze preequilibrium proton exchange at the nucleophilic carbon center at a rate consistent with the intermediate involvement of the conjugate base in the condensation reaction (6). The reaction is formally electrophilic substitution of a carbonyl carbon for a proton at the a-carbon atom of the enamine. Stereochemical studies have shown that the proton and carbonyl bind to the same face of the enamine carbon (carbanionic center) (Scheme 7) (7). 

This discrepancy between enzyme turnover and proton exchange presented a puzzle. Enzymes with turnover rates larger than 10 s like carbonic anhydrase and acetylcholinesterase, were dubbed impossible enzymes This is still sometimes reiterated with respect to these and other systems. However most biochemical reactions are studied in the presence of buffers. If the reaction of carbonic anhydrase is studied in the presence of 10 mM buffer with p =8, then the rate constant for proton exchange... 

If a carbanion is thermodynamically accessible, but is subject to rapid quenching by internal return of C02 in the case of decarboxylation, or by a proton in carboxylation, or in a hydrogen/deuterium exchange reaction, then the carbanionic intermediate off the enzyme would give the appearance of greater basicity than its thermodynamic value would predict. The localized character of the carbanion at the 6-position of UMP requires that the proton that is removed by a base in solution initially remains closely associated, and therefore, to a great extent be transferred to the carbanion. This reduces the rate of exchange and creates a discrepancy between kinetic and thermodynamic acidities. 

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