INHIBITION OF IMPDH BY MYCOPHENOLIC-ACID - DISSECTION OF FORWARD AND REVERSE PATHWAYS USING CAPILLARY ELECTROPHORESIS

The objective of this work was to contribute to the understanding of m echanisms for IMPDH inhibition. We over-expressed hamster type II IMPD H in Escherichia coli, purified the protein to apparent homogeneity, a nd used capillary electrophoresis to quantify enzyme turnover events a ccompanying inhibition by mycophenolic acid (MPA). We dissected two co nvergent pathways leading to MPA-inhibition; a rapid ''forward'' pathw ay beginning with substrates and linked to enzyme catalysis, and a slo wer ''reverse'' pathway apparently not involving catalysis. MPA-inhibi tion occurred rapidly in the forward direction by interrupting the enz yme turnover cycle, after IMP and NAD(+) binding, after hydride transf er, and after NADH release. Slow inhibition, without substrate turnove r, was achieved by incubating free enzyme with excess XMP and MPA. We propose that mycophenolic acid inhibits IMPDH by trapping a transient covalent product of the hydride transfer reaction (IMPDH similar to XM P) before a final hydrolysis step that precedes XMP and enzyme releas e in the forward reaction pathway, Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, ch emically defined complexes for structural studies. IMPDH samples inhib ited by MPA in the forward and reverse pathways yielded similar, high- quality crystals that are currently undergoing X-ray diffraction analy ses.