Sandbox Reserved 768: Difference between revisions

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== Phenylalanine Hydroxylase Mechanism of Action ==

PAH, belonging to the oxidoreductase enzyme class, acts on paired donors with pteridine being a donor and first incorporates one atom of molecular oxygen into the aromatic ring of phenylalanine. Then, it reduces the second oxygen atom to water using the two electrons that are supplied by the BH4 cofactor. BH4 is also hydroxylated at each turnover to produce pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme. 4a-OHBH4 is dehydrated and reduced back to BH4 by the action of the enzyme pterin carbinolamine dehydratase. <ref name= "flydal"/>.

PAH, belonging to the oxidoreductase enzyme class, acts by oxidizing/reducing its substrate. Specifically, PAH acts on paired donors with pteridine being a donor and first incorporates one atom of molecular oxygen into the aromatic ring of phenylalanine. <ref name= "pdb"/>. Then, it reduces the second oxygen atom to water using the two electrons that are supplied by the BH4 cofactor. BH4 is also hydroxylated at each turnover to produce pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme. 4a-OHBH4 is dehydrated and reduced back to BH4 by the action of the enzyme pterin carbinolamine dehydratase. <ref name= "flydal"/>.

[[Image:PAH mechanism of action.jpg]]

== Kinetics Overview ==

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>.

Sandbox Reserved 768: Difference between revisions

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@@ Line 15: / Line 15: @@
 == Phenylalanine Hydroxylase Mechanism of Action ==
-PAH, belonging to the oxidoreductase enzyme class, acts on paired donors with pteridine being a donor and first incorporates one atom of molecular oxygen into the aromatic ring of phenylalanine. Then, it reduces the second oxygen atom to water using the two electrons that are supplied by the BH4 cofactor. BH4 is also hydroxylated at each turnover to produce pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme. 4a-OHBH4 is dehydrated and reduced back to BH4 by the action of the enzyme pterin carbinolamine dehydratase. <ref name= "flydal"/>.
+PAH, belonging to the oxidoreductase enzyme class, acts by oxidizing/reducing its substrate. Specifically, PAH acts on paired donors with pteridine being a donor and first incorporates one atom of molecular oxygen into the aromatic ring of phenylalanine. <ref name= "pdb"/>. Then, it reduces the second oxygen atom to water using the two electrons that are supplied by the BH4 cofactor. BH4 is also hydroxylated at each turnover to produce pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme. 4a-OHBH4 is dehydrated and reduced back to BH4 by the action of the enzyme pterin carbinolamine dehydratase. <ref name= "flydal"/>.
+[[Image:PAH mechanism of action.jpg]]
+== Kinetics Overview ==
+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>.