Sandbox Reserved 768: Difference between revisions

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Formation of the productive PAH−BH4−phenylalanine complex begins with the rapid binding of BH4 (Kd = 65 μM). Subsequently, phenylalanine is added to the binary complex to form the productive ternary complex (Kd = 130 μM). This second step is approximately 10-fold slower. Both substrates are able to bind to the free enzyme form to produce inhibitory binary complexes. Molecular oxygen rapidly binds to the formed productive ternary complex; which is then followed by formation of an unidentified intermediate. This intermediate can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s−1. Formation of the 4a-OHBH4 and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines kcat. <ref> Roberts, Kenneth, Jorge Pavon, and Paul Fitzpatrich. "Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single-Turnover Experiments." Biochemistry 52(2013): 1062-1073. Web. </ref>.

== Phenylalanine Hydroxylase Structure ==

Although the full-length structure of mammalian PAH has not yet been clearly known, a large part of it has been identified. <ref name= "flydal"/>. It was solved by means of crystallizing the protein (at pH=7) to perform X-ray chromatography using molecular replacement. The search model used was based on the crystal structure of tyrosine hydroxylase because of the similarity between the two enzymes. <ref name=" fusetti"/> Fusetti, Fabrizia, Heidi Erlandsen, Torgeir Flatmark, and Raymond Stevens. "Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria." The Journal of Biological Chemistry 273.27 (1998): 16962-16967. Web. </ref>. The R-factor recorded was 0.251 and the mean B (or temperature) value was 33.0. <ref name= "pdb"/>.

'''Structure Revealed'''

The monomeric unit of PAH is composed of three sites: an N-terminal regulatory

domain (residues 1–110), a core catalytic domain

(residues 111–410), and a C-terminal oligomerization or tetramerization domain

(residues 411–452). <ref name= "flydal"/>.

The N-terminal regulatory domain is a regulatory module

present in several proteins, which functions in the dimerization and binding of amino acids. It is flexibly attached to the catalytic domain via a hinge region (Arg111–Thr117). <ref name= "flydal"/>.

The catalytic domain contains the active site of the enzyme. It is composed of 13 α-helices and 8 β-strands <ref name= "fusetti"/> and houses the binding sites for the nonheme iron atome, the cofactor, and substrate. The iron binds to two histidines (His285 and His290 in hPAH) and a glutamate (Glu330) in the deep cleft in the core of each monomer. <ref name=: "flydal"/>.

The C-terminal oligomerization or tetramerization domain begins with an antiparallel-sheet

(residues 411–414, 421–424)

and is formed by a C-terminal “arm” consisting of two β-strands, forming a β-ribbon, and a 40 Å long α-helix. This C-terminal arm extends over an adjacent monomer, thus bringing the four helices (one from each monomer) into a closely packed anti-parallel coiled-coil motif in the center of the structure (as can be seen in the tetramer structure above). <ref name= "fusetti"/>.

Sandbox Reserved 768: Difference between revisions

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@@ Line 24: / Line 24: @@
 Formation of the productive PAH−BH4−phenylalanine complex begins with the rapid binding of BH4 (Kd = 65 μM). Subsequently, phenylalanine is added to the binary complex to form the productive ternary complex (Kd = 130 μM). This second step is approximately 10-fold slower. Both substrates are able to bind to the free enzyme form to produce inhibitory binary complexes. Molecular oxygen rapidly binds to the formed productive ternary complex; which is then followed by formation of an unidentified intermediate. This intermediate can be detected as a decrease in absorbance at 340 nm, with a rate constant of 140 s−1. Formation of the 4a-OHBH4 and Fe(IV)O intermediates is 10-fold slower and is followed by the rapid hydroxylation of the amino acid. Product release is the rate-determining step and largely determines kcat. <ref> Roberts, Kenneth, Jorge Pavon, and Paul Fitzpatrich. "Kinetic Mechanism of Phenylalanine Hydroxylase: Intrinsic Binding and Rate Constants from Single-Turnover Experiments." Biochemistry 52(2013): 1062-1073. Web. </ref>.
+== Phenylalanine Hydroxylase Structure ==
+Although the full-length structure of mammalian PAH has not yet been clearly known, a large part of it has been identified. <ref name= "flydal"/>. It was solved by means of crystallizing the protein (at pH=7) to perform X-ray chromatography using molecular replacement. The search model used was based on the crystal structure of tyrosine hydroxylase because of the similarity between the two enzymes. <ref name=" fusetti"/> Fusetti, Fabrizia, Heidi Erlandsen, Torgeir Flatmark, and Raymond Stevens. "Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria." The Journal of Biological Chemistry 273.27 (1998): 16962-16967. Web. </ref>. The R-factor recorded was 0.251 and the mean B (or temperature) value was 33.0. <ref name= "pdb"/>.
+'''Structure Revealed'''
+The monomeric unit of PAH is composed of three sites: an N-terminal regulatory
+domain (residues 1–110), a core catalytic domain
+(residues 111–410), and a C-terminal oligomerization or tetramerization domain
+(residues 411–452). <ref name= "flydal"/>.
+The N-terminal regulatory domain is a regulatory module
+present in several proteins, which functions in the dimerization and binding of amino acids. It is flexibly attached to the catalytic domain via a hinge region (Arg111–Thr117). <ref name= "flydal"/>.
+The catalytic domain contains the active site of the enzyme. It is composed of 13 α-helices and 8 β-strands <ref name= "fusetti"/> and houses the binding sites for the nonheme iron atome, the cofactor, and substrate. The iron binds to two histidines (His285 and His290 in hPAH) and a glutamate (Glu330) in the deep cleft in the core of each monomer. <ref name=: "flydal"/>.
+The C-terminal oligomerization or tetramerization domain begins with an antiparallel-sheet
+(residues 411–414, 421–424)
+and is formed by a C-terminal “arm” consisting of two β-strands, forming a β-ribbon, and a 40 Å long α-helix. This C-terminal arm extends over an adjacent monomer, thus bringing the four helices (one from each monomer) into a closely packed anti-parallel coiled-coil motif in the center of the structure (as can be seen in the tetramer structure above). <ref name= "fusetti"/>.