ENZYPLOTW: A USEFUL TOOL TO CALCULATE ENZYME KINETIC PARAMETERS

F.A Leone, R.P.M. Furriel, I.A. Borin, J.C. McNamara

Resumo


Assays  of  enzyme  activity  are  among  the  most  frequently  employed  procedures  in  biochemistry.  They  are used to estimate the amount  of a given enzyme in a cell or tissue, to quantify kinetic parameters or to investigate a catalytic  mechanism.  The purpose of enzyme  kinetics  obtained  under steady-state conditions is  to estimate  KM and VM values by fitting initial rate (v) and substrate concentration (S) values to the Michaelis-Menten equation, permitting a convenient graphical representation and an accurate estimation of KM and VM. However, there is no ideal assay for any  particular  enzyme  and,  since  the  assay  of enzyme  activity  is  essentially a  kinetic  measurement,  various  pitfalls await  the unwary investigator.  Despite  difficulties  consequent to intrinsic  enzyme  properties,  kinetic  parameters  can be  estimated  accurately  when  steady-state  conditions  (<10%  variation  in  substrate  concentration  during  activity assay) and initial rate measurements (catalytic enzyme concentration) are guaranteed.EnzyplotW  is  an application developed for  use  on any  Intel-based  computer running  Windows 98 or  later, and uses a non-linear  regression method to fit steady-state kinetic data for Michaelian enzymes. EnzyplotW  can be employed not only for educational purposes but also in routine laboratory work, and includes three important features: teaching students how to obtain and recognize sound data for the best estimation of kinetic parameter values; aid the investigator  at the bench  in gauging  the limitations  of the various  equivalent plots  of the Michaelis-Menten  equation used to estimate KM and VM; and allow the student to manipulate the mathematical formula of the corresponding plot to  comprehend  the  qualitative  and  quantitative  issues  that  govern  the  relationship  between  substrate  concentration and reaction rate.

Palavras-chave


ENZYME KINETIC, EnzyplotW, Michaelian enzymes.

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DOI: https://doi.org/10.16923/reb.v4i3.56

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