Spectroscopic and Kinetic Studies of PKU−Inducing Mutants of Phenylalanine Hydroxylase: Arg158Gln and Glu280Lys

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Abstract
Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent, nonheme iron enzyme that catalyzes the hydroxylation of l-Phe to l-Tyr in the rate-limiting step of phenylalanine catabolism. This reaction is tightly coupled in the wild-type enzyme to oxidation of the tetrahydropterin cofactor. Dysfunction of PAH activity in humans leads to the disease phenylketonuria (PKU). We have investigated two PKU-inducing mutants, Arg158Gln and Glu280Lys, using kinetic methods, magnetic circular dichrosim (MCD) spectroscopy, and X-ray absorption spectroscopy (XAS). Analysis of the products produced by the mutant enzymes shows that although both oxidize pterin at more than twice the rate of wild-type enzyme, these reactions are only ∼20% coupled to production of l-Tyr. Previous MCD and XAS studies had demonstrated that the resting FeII site is six-coordinate in the wild-type enzyme and converts to a five-coordinate site when both l-Phe and reduced pterin are present in the active site. Although the Arg158Gln mutant forms the five-coordinate site when both cosubstrates are bound, the FeII site of the Glu280Lys mutant remains six-coordinate. These results provide insight into the PAH reaction and disease mechanism at a molecular level, indicating that the first step of the mechanism is formation of a peroxy-pterin species, which subsequently reacts with the FeII site if the pterin is properly oriented for formation of an Fe−OO-pterin bridge and an open coordination position is available on the FeII.

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