Recombinant human inosine monophosphate dehydrogenase type I and type II proteins. Purification and characterization of inhibitor binding.
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 kcat values of 1.8 and 1.4 sec-1, respectively, with Km values for IMP of 14 and 9 microM and Km values for NAD of 42 and 32 microM. The two enzymes were inhibited competitively by the immunosuppressive agent mizoribine 5'-monophosphate (MMP) with Ki values of 8 and 4 nM and inhibited uncompetitively by mycophenolic acid with Ki values of 11 and 6 nM. The association of MMP to either isozyme, as monitored by fluorescence quenching, was relatively slow with kon values of 3-8 x 10(4) M-1 sec-1 and koff values of 3 x 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.
Hager
P W
PW
Department of Pharmacology, University of North Carolina at Chapel Hill 27514, USA.
Collart
F R
FR
Huberman
E
E
Mitchell
B S
BS
D001667:Binding, Competitive; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D009173:Mycophenolic Acid / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification; D012263:Ribonucleosides / Q000494:pharmacology
D007166:Immunosuppressive Agents; D007527:Isoenzymes; D011994:Recombinant Proteins; D012263:Ribonucleosides; C010052:mizoribine; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid