Recombinant human inosine monophosphate dehydrogenase type I and type II proteins: Purification and characterization of inhibitor binding

Abstract

Inosine monophosphate dehydrogenase (IMPDH) activity results from the expression of two separate genes, and the resulting proteins (type I and type II) are 84% identical at the amino acid level. Although the type II mRNA is expressed at higher levels in proliferating cells, both mRNAs, and by extrapolation both proteins, are present in normal and malignant cells. Since IMPDH is an important target for the development of drugs with both chemotherapeutic and immunosuppressive activity, we have compared the kinetic and physical properties of the two human enzymes expressed in and purified from Escherichia coli. Type I and II IMPDH had k cat values of 1.8 and 1.4 sec −1, respectively, with K m values for IMP of 14 and 9 μM and K m values for NAD of 42 and 32 μM. The two enzymes were inhibited competitively by the immunosuppressive agent mizoribine 5′-monophosphate (MMP) with K i values of 8 and 4 nM and inhibited uncompetitively by mycophenolic acid with K i values of 11 and 6 nM. The association of MMP to either isozyme, as monitored by fluorescence quenching, was relatively slow with k on values of 3–8 × 10 4 M −1 sec −1 and k off values of 3 × 10 −4 sec −1 (half-lives of 36–43 min). Thus, MMP is a potent, tight-binding competitive inhibitor that does not discriminate between the two IMPDH isozymes.