Mechanistic inferences from the crystal structure of fumarylacetoacetate hydrolase with a bound phosphorus-based inhibitors

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield fumarate and acetoacetat e 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 m echanism involving a catalytic metal ion, a Glu/His catalytic dyed, and a c harged oxyanion hole was previously proposed based on recently determined F AH crystal structures. Here we report the development and characterization of an FAH inhibitor, 4-(hydroxymethylphosphinoyl)-3-oxo-butanoic acid (HMPO BA), that competes with the physiological substrate with a K-i of 85 muM. T he crystal structure of FAH complexed with HMPOBA refined at 1.3-Angstrom r esolution reveals the molecular basis for the competitive inhibition, suppo rts the proposed formation of a tetrahedral alkoxy transition state interme diate during the FAH catalyzed reaction, and reveals a Mg2+ bound in the en zyme's active site. The analysis of FAH structures corresponding to differe nt catalytic states reveals significant active site sidechain motions that may also be related to catalytic function. Thus, these results advance the understanding of an essential catabolic reaction associated with a fatal me tabolic disease and provide insight into the structure-based development of FAH inhibitors.