Mutations within the second coordination sphere of catalytic iron (II) in human phenylalanine hydroxylase (hPAH) that affect the environment and electronic geometry of iron (II) are implicated in its mechanism, substrate binding and specificity.

摘要

Phenylalanine hydroxylase (PAH) is a mononuclear non-heme iron enzyme that catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and reduced pterin. X-ray studies established the coordination around the iron metal center and point to significant interactions within the second coordination sphere where L-Phe and BH4 bind. Such interactions implicated in the hydrogen bonding effect of human phenylalanine hydroxylase (hPAH) involve Y325, E286, Y138, R270 and D282.; Y325F hPAH mutant enzyme showed similar kinetics, thermal stability, and oligomerization profile as wild-type protein. The possibility of in vivo post-translational hydroxylation that would restore the activity of hPAH was examined by mass spectrometry on trypsin digested full-length (1--452) hPAH Y325F point mutant. The amino acid tags obtained by ESI-MS/MS confirmed the presence of a F325 in the peptide corresponding to the doubly charged precursor ion at m/z 916.4 and its hydroxylated counterpart in the peptide corresponding to m/z 924.4. Furthermore, the point mutation Y325A resulted in an enzyme that was totally inactive, and did not display any evidence of hydroxylation.; To address the effect of substrate binding on the coordination environment of catalytic iron, NO was reacted with several mutants (Y325F, E286A, Y138F, R270S, R270K, and D282) to yield iron-nitrosyl complexes to study by EPR and circular dichroism. The mutant proteins retained their a-helical fold that is responsible for their overall secondary structure. The anaerobic addition to Y325F, E286A, and Y138F of 6MPH4 and L-Phe either before or after exposure of the enzyme to NO resulted in greater rhombic distortion and the formation of at least two S = 3/2 species. The conversion of the iron environment to a mixed rhombic signal was similar to that observed with wild-type enzyme. To reconcile a lack of hydroxylation with intact iron geometry in mutant E286A, we speculate that during the catalytic turnover, the peroxypterin is not properly oriented to form the Fe-O-O-pterin bridge which could lead to the Fe (IV) oxo intermediate that acts as the hydroxylating agent. Meanwhile, R270S, R270K and D282S enzymes expressed as inactive forms with low Fe content that make it impossible to observe a meaningful distortion signal.