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## Brazilian Journal of Pharmaceutical Sciences

*versão On-line* ISSN 2175-9790

#### Resumo

ULUDAG-DEMIRER, Sibel; DURAN, Jorge e TANNER, Robert D.. **Estimating the turnover number in enzyme kinetic reactions using transient and stationary state data**.* Braz. J. Pharm. Sci.* [online]. 2009, vol.45, n.4, pp.635-642.
ISSN 2175-9790. http://dx.doi.org/10.1590/S1984-82502009000400005.

Substrate and product concentration data obtained by simulating enzyme-substrate reaction rate equations were used to test two proposed kinetic rate constant estimation techniques in this study. In the first technique, the turnover number, k_{3}, was calculated using early transient time domain data, which are difficult to obtain experimentally. The technique used an iterative approach to calculate k_{3} with a pair of data and the value of k_{3} could be retrieved with 35% error. The second technique calculated k_{3} using stationary domain data and the value of k_{3} could be retrieved with less than 5% error. This second technique also offered internal consistency in the calculation of k_{3} by calculating k_{3} both from the intercept and the slope of the linear plot derived in this study. A series of sensitivity analyses was conducted to understand the robustness of the second technique in estimating k_{3} from simulated data to the changes in the reaction rate constants (k_{1}, k_{2}, and k_{3}) and the initial concentration of enzyme used for simulation. It was found that the second technique generally worked well in the estimation of k_{3} except for the simulated data for fast substrate conversions such as in the large k_{3} and [E]_{0} cases . This latter method, thus, shows promise for the use of late time experimental substrate/product concentration data to obtain k_{3}. Exclusively using late time data avoids the need for difficult and expensive rapid early time measurement techniques for estimating k_{3}. Once a reasonable estimate for k_{3} is obtained, the initial enzyme value can easily be determined from the maximum velocity constant established from fitting the Michaelis-Menten or Briggs-Haldane equations to substrate and product stationary state domain (late time) data. While the first technique can estimate k_{3} with only one point in the transient domain, it is suggested that the second method generally be favored since it only requires late-time stationary domain data and appears to be more accurate.

**Palavras-chave
:
**Enzyme; Kinetics [rate constant]; Enzyme reactions.