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Methods of determining rate constants in single-substrate-single-product enzyme reactions. Use of induced transport: limitations of product inhibition.

ABSTRACT: 1. The calculation of the rate constants from steady-state kinetics of a single-substrate-single-product enzyme reaction in which there is an isomerization of the enzyme is described. 2. It is shown that even with the use of isotopically labelled substrates a set of solutions for the constants is obtained rather than a unique solution. However, limits are derived within which they must lie. 3. The most appropriate observations to determine the rate constants are measurements of V(max.) and K(m) for both substrate and product, and measurement of the degree of countertransport in an induced-transport test. 4. Experimental procedures for induced-transport tests and the quantitative interpretation of the results obtained are discussed. 5. Product inhibition is shown to be an ambiguous and imprecise means of determining the rate constants. Further, the absence of a [substrate]x[product] term in the denominator of the steady-state rate equation does not necessarily mean that the isomerization of the enzyme is rapid, since the term also disappears when the isomerization is very slow. 6. Similar considerations apply to carrier mechanisms.

SUBMITTER: Britton HG

PROVIDER: S-EPMC1177694 | biostudies-other | 1973 Jun

REPOSITORIES: biostudies-other

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Project description:The forward and reverse H + CO ↔ HCO reactions are important for combustion chemistry and have been studied from a wide variety of theoretical and experimental techniques. However, most of the chemical kinetic investigations concerning these processes are focused on low pressures or fall-off regions. Hence, a high-level electronic structure treatment was employed here in order to provide accurate rate constant values for these reactions at the high-pressure limit along temperatures from 50 to 4000 K. In relative terms, the variational effects on rate constants are shown to be almost as important at high temperatures as quantum tunneling corrections at the lowest temperatures investigated. The activation energies fitted by using modified and traditional Arrhenius' equations for the forward rate constants from 298 to 2000 K are, respectively, equal to 2.64 and 3.89 kcal mol-1, while similar fittings provided, respectively, 1.96 and 3.22 kcal mol-1, considering now forward rate constants from a temperature range of 298-373 K. This last activation energy determination (3.22 kcal mol-1) is in better agreement with the commonly referenced experimental value of 2.0 ± 0.4 kcal mol-1, also obtained from traditional fittings in the range 298-373 K, than the value attained from a broader temperature range fitting (3.89 kcal mol-1). However, some additional care must be considered along these comparisons once the experimental reaction rate measurements have been done for the trimolecular H + CO + M → HCO + M reaction instead. Anyway, the usage of appropriate temperature ranges is fundamental for reliable activation energy comparisons, although the remaining deviation between theory and experiment is still large and is possibly caused by the different pressure regimes assessed in each case. Finally, we roughly estimated that the high-pressure limit for the HCO decomposition into H and CO can be achieved along temperatures ranging from ∼246 and ∼255 K downward, respectively, at pressures of 1.1 and 9.6 atm, although further experiments should be carried out in order to improve these estimates. On the other hand, pressures larger than 9.8 × 104 atm are required for the aforementioned dissociation reaction to attain the high-pressure limit at 700 K. Therefore, the rate constants determined here are probably applicable in combustion studies at lower temperatures.

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Methods of determining rate constants in single-substrate-single-product enzyme reactions. Use of induced transport: limitations of product inhibition.

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