Enzyme inhibition assays using fluorescence correlation spectroscopy: A new algorithm for the derivation of k(cat)/K-M and K-i values at substrate concentrations much lower than the Michaelis constant

A new mathematical formalism is deduced which allows for the calculation of the k(cat) over K-M ratio based on measurements of the enzyme kinetics wit h substrate concentrations much lower than K-M. The equations are also appl ied on the action of an inhibitor on enzyme activity yielding the binding c onstant, K-i, of an inhibitor molecule. For practical evaluation of the new theoretical approach, the enzymatic reaction of CD45 phosphatase was used as a well-characterized model system with known inhibitors for testing the K-i value determination scheme. The k(cat)/K-M ratio was calulated to be 4. 7 x 10(5) M-1 s(-1), the K-i of the inhibitor molecule PKF52-524 was estima ted to be (1-2) x 10(-7) M and the association rate of the inhibitor PKF52- 524 to CD45 phosphatase was estimated to be 59 M-1 s(-1).