What students must know about the determination of enzyme kinetic parameters
ABSTRACT 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.
Article: Features: Problem Based LearningBiochemistry and Molecular Biology Education 12/2008; 28(6):330 - 331. · 0.70 Impact Factor
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ABSTRACT: The commonly used methods for bioconversion kinetic parameter identification are linear plotting and nonlinear regression. However, linear plotting methods generally require considerable experimentation, and nonlinear regression can lead to “local optimization” because the obtained parameters depend heavily on given initial values. In this article, a new and reliable nonlinear regression-based approach to bioconversion kinetic parameter estimation is reported. By obtaining preliminary values of kinetic parameters on a step-by-step basis, the number of estimated parameters in each step can be reduced to 3 or 4. These preliminary values can then be used as initial guesses for the final parameter estimation via nonlinear regression. Compared with the linear plotting method, the proposed approach can significantly reduce the number of experiments required for kinetic parameter estimation. The transketolase catalyzed synthesis of 1,3-dihydroxypentan-2-one from propionaldehyde and β-hydroxypyruvate is used as an experimental example to illustrate the approach. © 2008 American Institute of Chemical Engineers AIChE J, 2008AIChE Journal 06/2008; 54(8):2155 - 2163. · 2.49 Impact Factor
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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. · 0.70 Impact Factor