Spectroscopic and kinetic studies of PKU-inducing mutants of phenylalanine hydroxylase: Arg158Gln and Glu280Lys.
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 approximately 20% coupled to production of L-Tyr. Previous MCD and XAS studies had demonstrated that the resting Fe(II) 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 Fe(II) 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 Fe(II) site if the pterin is properly oriented for formation of an Fe-OO-pterin bridge and an open coordination position is available on the Fe(II).
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Jyllian N
JN
Kemsley
Erik C
EC
Wasinger
Supratim
S
Datta
Natasa
N
Mitić
Tara
T
Acharya
Britt
B
Hedman
John P
JP
Caradonna
Keith O
KO
Hodgson
Edward I
EI
Solomon
D015502:Absorptiometry, Photon; D001665:Binding Sites; D002942:Circular Dichroism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010651:Phenylalanine Hydroxylase / Q000235:genetics* / Q000378:metabolism; D010661:Phenylketonurias / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D005296:Ferrous Compounds; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010651:Phenylalanine Hydroxylase