The Use of Inosine 5′-Monophosphate Dehydrogenase (IMPDH) in the Development of a New Liquid Homogeneous Enzyme Immunoassay Technology

Abstract

The objective of the present study was the development of a quantitative liquid homogeneous immunoassay specific for theophylline based on the specific uncompetitive inhibition of inosine 5′-monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). This was accomplished by covalent coupling of theophylline to MPA to form a theophylline-MPA conjugate (Fig. 1⇓ ). IMPDH (EC 1.1.1.205) (1) catalyzes the NAD-dependent oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP). The enzyme follows an ordered Bi-Bi reaction sequence of substrate and cofactor binding and product release. In the first step, IMP binds to IMPDH, followed by the binding of the cofactor NAD. After IMP oxidation, the reduced cofactor, NADH, is released from IMPDH, followed by the release of XMP. To monitor the reaction, the rate of NADH formation is measured at 340 nm. Uncompetitive inhibition occurs when MPA combines with the IMPDH-XMP complex at the active site of the enzyme to form IMPDH-XMP-MPA complex, which is unable to release XMP. IMPDH inhibition depends only on the concentration of MPA because of the uncompetitive nature of inhibition by MPA. Thus, the greater the concentration of MPA inhibitor, the greater the inhibition of the enzyme. An uncompetitive inhibitor of IMPDH inhibits by binding at the active site of the enzyme and does not compete with IMP …