Enzyme Inhibition Assays Using Fluorescence Correlation Spectroscopy: A New Algorithm for the Derivation of kcat/KM and Ki Values at Substrate Concentrations Much Lower than the Michaelis Constant
- 1 October 2000
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 39 (43) , 13261-13268
- https://doi.org/10.1021/bi000057y
Abstract
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 with substrate concentrations much lower than K(M). The equations are also applied on the action of an inhibitor on enzyme activity yielding the binding constant, 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 estimated 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).Keywords
This publication has 6 references indexed in Scilit:
- Enzyme KineticsPublished by Cambridge University Press (CUP) ,1994
- CD45: An Emerging Role as a Protein Tyrosine Phosphatase Required for Lymphocyte Activation and DevelopmentAnnual Review of Immunology, 1994
- Substrate inhibition or activation kinetics of the β-galactosidase from the extreme thermoacidophile archaebacterium Caldariella acidophilaBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1990
- Substrate aggregation and cooperative enzyme kinetics: Consideration of enzyme access with large aggregatesJournal of Theoretical Biology, 1985
- Conformational specificity of chymotrypsin toward proline-containing substratesBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1984
- XXXVI.—Enzyme actionJournal of the Chemical Society, Transactions, 1902