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Originally published In Press as doi:10.1074/jbc.M007621200 on January 11, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15284-15291, May 4, 2001
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Mechanistic Inferences from the Crystal Structure of Fumarylacetoacetate Hydrolase with a Bound Phosphorus-based Inhibitor*

Raynard L. BatemanDagger §, P. Bhanumoorthy, John F. Witte§, Ronald W. McClard§, Markus GrompeDagger , and David E. Timm||

From the Dagger  Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201, the § Department of Chemistry, Reed College, Portland, Oregon 97202, and the  Department of Biochemistry, Indiana University, Indianapolis, Indiana 46202

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetate as the final step of Phe and Tyr degradation. This unusual reaction is an essential human metabolic function, with loss of FAH activity causing the fatal metabolic disease hereditary tyrosinemia type I (HT1). An enzymatic mechanism involving a catalytic metal ion, a Glu/His catalytic dyad, and a charged oxyanion hole was previously proposed based on recently determined FAH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPOBA), that competes with the physiological substrate with a Ki of 85 µM. The crystal structure of FAH complexed with HMPOBA refined at 1.3-Å resolution reveals the molecular basis for the competitive inhibition, supports the proposed formation of a tetrahedral alkoxy transition state intermediate during the FAH catalyzed reaction, and reveals a Mg2+ bound in the enzyme's active site. The analysis of FAH structures corresponding to different catalytic states reveals significant active site side-chain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal metabolic disease and provide insight into the structure-based development of FAH inhibitors.

* This work was supported by NIDDK, National Institutes of Health (NIH) grants (to D. E. T. and M. G.), by an NIGMS, NIH grant (to R. W. M.), and by grants from the Indiana Affiliate of the American Heart Association and the Grace M. Showalter Research Trust Fund (to D. E. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1HYO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

|| To whom correspondence should be addressed: Dept. of Biochemistry, Indiana University, Indianapolis, IN 46202. Tel.: 317-274-1551; Fax: 317-274-4686; E-mail: dtimm@iupui.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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