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 mechanisms for IMPDH inhibition. We over-expressed hamster type II IMPDH in Escherichia coli, purified the protein to apparent homogeneity, and used capillary electrophoresis to quantify enzyme turnover events accompanying inhibition by mycophenolic acid (MPA). We dissected two convergent pathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates and linked to enzyme catalysis, and a slower "reverse" pathway apparently not involving catalysis. MPA-inhibition occurred rapidly in the forward direction by interrupting the enzyme turnover cycle, after IMP and NAD+ binding, after hydride transfer, and after NADH release. Slow inhibition, without substrate turnover, 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 approximately XMP*) before a final hydrolysis step that precedes XMP and enzyme release in the forward reaction pathway. Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, chemically defined complexes for structural studies. IMPDH samples inhibited by MPA in the forward and reverse pathways yielded similar, high-quality crystals that are currently undergoing X-ray diffraction analyses.
Fleming
M A
MA
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211, USA. fleming@vpharm.com
Chambers
S P
SP
Connelly
P R
PR
Nimmesgern
E
E
Fox
T
T
Bruzzese
F J
FJ
Hoe
S T
ST
Fulghum
J R
JR
Livingston
D J
DJ
Stuver
C M
CM
Sintchak
M D
MD
Wilson
K P
KP
Thomson
J A
JA
D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D003460:Crystallization; D017931:DNA Primers / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009173:Mycophenolic Acid / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D007291:Inosine Monophosphate; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid