Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose–response curves

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Abstract
A statistical treatment of steady-state enzyme kinetic measurements is described that allows for depletion of free substrate or free inhibitor concentrations owing to significant binding to the enzyme. Vmax., Km or Ki, enzyme concentration, the concentration of substrate or inhibitor required for a half-maximal effect and standard errors of these parameters can be calculated from dose–response measurements; the concentration of each component of the system may be estimated also. The statistically best values of the parameters are used to convert dose–response curves into convenient linear forms. The method is applied to dose–response measurements of hydroxyquinoline N-oxide inhibition of bacterial respiration and aminopterin inhibition of dihydrofolate reductase. Two FORTRAN programs for this method have been deposited as Supplementary Publication no. SUP 50019 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973) 131, 5.