A simple method for calculating Km and V from a single enzyme reaction progress curve

Abstract

A method of calculating Km and V from a single reaction progress curve is presented. The integrated Michaelis-Menten equation Vt = So + Km 1n(So/S), may be rearranged to the form 1/v = 1/V + Km/VS, where v = (Si - Sj)/deltat = deltaS/deltat and S = (Si - Sj)/ln(Si/Sj) or S is approximated by (Si + Sj)/2.Si and Sj represent substrate concentrations at two points along a reaction progress curve separated by the time interval, deltat. The error resulting from the approximation depends on the magnitude of deltaSi/Si; when deltaSi/Si less than 0.3, the error is insignificant; when deltaSi/Si greater than 0.3, the error becomes significant. Procedures are presented to correct this error. Simulated data and application to the direct spectrophotometric assay of AMP aminohydrolase and the lactate dehydrogenase coupled assay of pyruvate kinase are provided. The method is recommended when routine Km and V values are desired. Compared to the initial rate method, it is faster, requires less substrate, and eliminates pipetting errors.