What student must know about the determination of enzyme kinetic parameters

University of Florence, Florens, Tuscany, Italy
Biochemical Education 06/2010; 27(2):87 - 91. DOI: 10.1016/S0307-4412(98)00301-X


After a brief overview of the limits of the graphical methods used to determine enzyme kinetic parameters, the paper shows the results of their application to simulated velocity data, influenced by experimental errors of increasing magnitude. The comparison indicates that the best method to evaluate Vmax and Km is nonlinear regression, even in the presence of constant relative error; whereas the double reciprocal plot should be avoided, unless used with a proper weighting factor. The paper also suggests a simple method to obtain computer-simulated velocity data, with which the student may get direct practise and experience.

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Available from: francesco.ranaldi@unifi.it fra3927, Oct 27, 2015
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    • "Determination of the required data for establishment of full kinetic models can be time and resource consuming, especially when using traditional linear data fitting methods [12] [39]. This problem is further accentuated for non-natural bioconversions used in the pharmaceutical industry that often exhibit strong substrate and product inhibition [54]. "
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    ABSTRACT: This work describes the establishment of a full kinetic model, including values of apparent kinetic parameters, for the whole cell E. coli mediated synthesis of the chiral amino-alcohol (2S,3R)-2-amino-1,3,4-butanetriol (ABT), using (S)-(−)-α-methylbenzylamine (MBA) as amino donor. The whole cell biocatalyst expressed the CV2025 ω-transaminase from Chromobacterium violaceum. Establishment of the most suitable reaction mechanism and determination of the complete forward and reverse kinetic parameter values for the reversible bioconversion where obtained using a hybrid methodology. This combined traditional initial rate experiments to identify a solution in the vicinity of the global minimum, with nonlinear regression methods to determine the exact location of the solution. The systematic procedure included selection and statistical evaluation of different kinetic models that best described the measured reaction rates and which ultimately provided new insights into the reaction mechanism; in particular the possible formation of a dead end complex between the amino donor and the cofactor enzyme complex. The hybrid methodology was combined with a microscale experimental platform, to significantly reduce both the number of experiments required as well as the time and material required for full kinetic parameter estimation. The equilibrium constant was determined to be 849, and the forward and reverse rate constants were found to be 97 and 13 min−1, respectively, which greatly favoured the asymmetric synthesis of chiral ABT. Using the established kinetic model, the asymmetric synthesis of ABT was simulated, and excellent agreement was found between the experimental and predicted data over a range of reaction conditions. A sensitivity analysis combined with various simulations suggested the crucial bottleneck of the reaction was the second half reaction of the ping pong bi–bi mechanism, in part due to the low Michaelis constant of substrate l-erythrulose (ERY). The toxicity of MBA towards the transaminase was identified as another major bottleneck. The kinetic model was useful to give early insights into the most appropriate bioconversion conditions, which can improve the rate and yield of ABT formation, as well as minimizing the toxicity and inhibition effects of the substrates and products. The systematic methodology developed here is considered to be generic and useful in regard to speeding up bioconversion process design and optimization.
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    • "The data in Table 2 can be used to determine the kinetic parameters of the purified enzymes. This can be done by using non-linear regression analysis of the Michaelis-Menten plots [6] or by obtaining linear double reciprocal plots [7] [8], as illustrated in Fig. 1 for the normal situation. By doing this, the values of K , and V,,, given in Table 4 are obtained for the different cases. "
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    ABSTRACT: Glucokinase is a key enzyme in carbohydrate metabolism in mammals. Particularly relevant is the involvement of the liver enzyme for the maintenance of blood glucose concentrations. In playing this key role, the occurrence of normal kinetic parameters of liver glucokinase is critical. Modification of Km and/or Vmax results in pathological situations occurring with hypo- or hyperglycemia. Based on this, an exercise is proposed in which different mice cell lines with abnormalities in glucose utilization are analyzed to determine the metabolic alteration at a molecular level. The student must determine the cause of abnormality by processing the kinetic data. Also, the characterization of different compounds that are reversible inhibitors of the enzyme from kinetic data is required to identify a drug that can correct the pathology in each case. One major objective of the problem is to learn about the meaning of kinetic parameters of enzymes, as well as the action of different types of reversible inhibitors. © 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved.
    Biochemistry and Molecular Biology Education 12/2008; 28(6):332 - 337. DOI:10.1111/j.1539-3429.2000.tb00186.x · 0.65 Impact Factor
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    • "For these reasons, the following article tenders an educational approach to teaching the theoretical principles and experimental concepts of slow-binding inhibition and to the development of students' practical laboratory skills with rapid kinetic techniques. Special emphasis is given to data analysis where we analyze experimental data from classical linear [1] [2] and newer nonlinear regression points of view [3]. The suggested course can be also instructed only partly, within economic, infrastructural, and space of time possibilities. "
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