Steady-state kinetics isotopic exchange

P Aminoacyl-tRNA synthetases Detection of intermediates by quenched flow, steady state kinetics, and isotope exchange... 

Segel, 1. H., Emyme Kinetics Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience (1975). This book starts at the same elementary level as Biochemical Calculations and progresses to the modern subjects of steady-state kinetics of mullireac-tant enzymes, allosteric enzymes, isotope exchange, and membrane transport. 

The Michaelis-Menten Formalism has been remarkably successful in elucidating the mechanisms of isolated reactions in the test tube. There are numerous treatments of this use of kinetics, and many of these provide a thoughtful critique of the potential pit falls. In short, reliable results can be obtained with steady-state methods if one is careful to follow the canons and if one remembers that several mechanisms may yield the same kinetic behavior. Isotope exchange, pre-steady state, and other transient or relaxation kinetic techniques, as well as various chemical and physical methods, also have been applied in conjunction with steady-state kinetic methods to dissect the elementary reactions within an enzyme-catalyzed reaction and to distinguish between various models (e.g., see Cleland, 1970 Kirschner, 1971 Segel, 1975 Hammes, 1982 Fersht, 1985). 

Differentiation between reaction mechanisms can be achieved by careful scmtiny of the K versus substrate concentration patterns (Fig. 7.4). The adage that a picture tells a thousand words is quite applicable in this instance. It is difficult to determine the mechanism of an enzyme-catalyzed reaction from steady-state kinetic analysis. The determination of the mechanism of an enzymatic reaction is neither a trivial task nor an easy task. The use of dead-end inhibitors and alternative substrates, study of the patterns of product inhibition, and isotope-exchange experiments... 

During steady-state isotopic transient kinetic analysis, the 12CO was switched to 13CO and the carbon-containing adsorbed and gas phase species were monitored in the IR as they exchanged from the 12C to the 13C label. Particular attention was made to those species that exchanged on a timescale similar to that of the exchange of the product C02, as that species could be a likely intermediate to the water-gas... 

Initial rate measurements, especially with alternative substrates and with a product or substrate analog as inhibitor, and measurements of the rate of isotope exchange at equilibrium, can give a great deal of information about mechanism, and in some cases allow estimates of individual velocity constants and dissociation constants. The results of such studies, which require little enzyme, are an essential basis for the proper interpretation, in relation to the overall catalytic reaction, of pre-steady-state studies and kinetic and thermodynamic studies of enzyme-coenzyme reactions in isolation. 

The steady-state rate equation for the random mechanism will also simplify to the form of Eq. (1) if the relative values of the velocity constants are such that net reaction is largely confined to one of the alternative pathways from reactants to products, of course. It is important, however, that dissociation of the coenzymes from the reactant ternary complexes need not be excluded. Thus, considering the reaction from left to right in Eq. (13), if k-2 k-i, then product dissociation will be effectively confined to the upper pathway this condition can be demonstrated by isotope exchange experiments (Section II,C). Further, if kakiB kik-3 -f- kikiA, then the rate of net reaction through EB will be small compared with that through EA 39). The rate equation is then the same as that for the simple ordered mechanism, except that a is now a function of the dissociation constant for A from the ternary complex, k-i/ki, as well as fci (Table I). Thus, Eqs. (5), (6), and (7) do not hold instead, l/4> < fci and ab/ a b < fc-i, and this mechanism can account for anomalous maximum rate relations. In contrast to the ordered mechanism with isomeric complexes, however, the same values for these two functions of kinetic coefficients would not be expected if an alterna-... 

The kinetics with all the substrates and isotope exchange studies are consistent with the preferred pathway mechanism described in Section II,B,4. With secondary alcohols, hydride transfer is slow, the steady-state concentration of the reactant ternary complex is large, and leak of NAD from this complex occurs thus, < a = Ak- /kki (Table I) and is different for different alcohols. With primary alcohols as substrates, hydride transfer and aldehyde dissociation are much faster than NADH dissociation. Under initial rate conditions, therefore, the ternary complex is not present in significant steady-state concentration, and dissociation of NAD from it does not occur to an appreciable extent thus, 4> = 1/ki, and like < o is the same for all primary alcohols. 

The following sections provide a kinetic analysis of the transient responses based on an atomic state for the chemisorbed oxygen, 0(s). We show that this approach allows us to account for the qualitative features of the results described above, the temperature dependence of the rate of isotope scrambling under steady-state conditions, and results ftom temperature programmed desorption (TPD) experiments performed at very low pressure. The steady-state exchange and TPD experiments are described in Sec. 3.1.3.. The kinetics of isotope exchange of O2 (gas) with oxide materials have been reviewed by Ceilings and Bouwmeester. Readers are referred to this work and references therein for a more comprehensive discussions of the mechanisms and kinetics involved in more complex systems. 

Highly specialized steady state studies have been carried out on selected enzymes to explore their reaction mechanisms (see for instance Dalziel, 1975, and the isotope exchange experiments mentioned above). However, the major contribution of such investigations is to the definition of those kinetic characteristics of enzymes which are of importance for an under-... 

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