Inhibition of IMPDH by Mycophenolic Acid: Dissection of Forward and Reverse Pathways Using Capillary Electrophoresis

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• 作者：Mark A. Fleming ; Stephen P. Chambers ; Patrick R. Connelly ; Elmar Nimmesgern ; Ted Fox ; Frank J. Bruzzese ; Stephen T. Hoe ; John R. Fulghum ; David J. Livingston ; Cameron M. Stuver ; Michael D. Sintchak ; K
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：June 4, 1996
• 年：1996
• 卷：35
• 期：22
• 页码：6990 - 6997
• 全文大小：373K
• 年卷期：v.35,no.22(June 4, 1996)
• ISSN：1520-4995

文摘

The objective of this work was to contribute to the understandingof mechanisms for IMPDHinhibition. We over-expressed hamster type II IMPDH inEscherichia coli, purified the protein toapparenthomogeneity, and used capillary electrophoresis to quantify enzymeturnover events accompanyinginhibition by mycophenolic acid (MPA). We dissected two convergentpathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates andlinked to enzyme catalysis, and aslower "reverse" pathway apparently not involving catalysis.MPA-inhibition occurred rapidly in theforward direction by interrupting the enzyme turnover cycle, after IMPand NAD⁺ binding, after hydridetransfer, and after NADH release. Slow inhibition, withoutsubstrate turnover, was achieved by incubatingfree enzyme with excess XMP and MPA. We propose that mycophenolicacid inhibits IMPDH by trappinga transient covalent product of the hydride transfer reaction(IMPDH~XMP*) before a final hydrolysisstep that precedes XMP and enzyme release in the forward reactionpathway. Understanding the ligandoccupancy of the protein has also proven important for producinghomogeneous, chemically definedcomplexes for structural studies. IMPDH samples inhibited by MPAin the forward and reverse pathwaysyielded similar, high-quality crystals that are currently undergoingX-ray diffraction analyses.