Kinetic Mechanism and Intrinsic Rate Constants for the Reaction of a Bacterial Phenylalanine Hydroxylase

The pterin-dependent aromatic amino acid hydroxylases are non-heme iron enzymes that catalyze the hydroxylation of the aromatic side chain of their respective substrates using an Fe^IVO intermediate. While the eukaryotic enzymes are homotetramers with complex regulatory properties, bacterial phenylalanine hydroxylases are monomers that lack regulatory domains. As a result, the bacterial enzymes are more tractable for mechanistic studies. Using single turnover methods, the complete kinetic mechanism and intrinsic rate constants for <i>Chromobacterium violaceum</i> phenylalanine hydroxylase have been determined with both tetrahydrobiopterin and 6-methyltetrahyropterin as substrates. In addition the kinetics of formation of the enzyme–pterin complex have been determined with the unreactive 5-deaza, 6-methyltetrahydropterin. For all three pterins, binding of phenylalanine and pterin occurs in random order with binding of the pterin first the preferred pathway. The reaction of the ternary enzyme–phenylalanine–tetrahydropterin complex can be described by a mechanism involving reversible oxygen binding, formation of an early intermediate preceding formation of the Fe^IVO, and rate-limiting product release

Mutagenesis of an Active-Site Loop in Tryptophan Hydroxylase Dramatically Slows the Formation of an Early Intermediate in Catalysis

Solution studies of the aromatic amino acid hydroxylases are consistent with the Fe^IVO intermediate not forming until both the amino acid and tetrahydropterin substrates have bound. Structural studies have shown that the positions of active-site loops differs significantly between the free enzyme and the enzyme-amino acid-tetrahydropterin complex. In tryptophan hydroxylase (TrpH) these mobile loops contain residues 124–134 and 365–371, with a key interaction involving Ile366. The I366N mutation in TrpH results in decreases of 1–2 orders of magnitude in the <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m values. Single turnover analyses establish that the limiting rate constant for turnover is product release for the wild-type enzyme but is formation of the first detectable intermediate <b>I</b> in catalysis in the mutant enzyme. The mutation does not alter the kinetics of NO binding to the ternary complex nor does it uncouple Fe^IVO formation from amino acid hydroxylation. The effects on the <i>k</i>_cat value of wild-type TrpH of changing viscosity are consistent with rate-limiting product release. While the effect of viscosity on the <i>k</i>_cat/<i>K</i>_O₂ value is small, consistent with reversible oxygen binding, the effects on the <i>k</i>_cat/<i>K</i>_m values for tryptophan and the tetrahydropterin are large, with the latter value exceeding the expected limit and varying with the identity of the viscogen. In contrast, the kinetic parameters of I366N TrpH show small changes with viscosity. The results are consistent with binding of the amino acid and pterin substrate to form the ternary complex being directly coupled to closure of loops over the active site and formation of the reactive complex. The mutation destabilizes this initial event

Phenylalanine Binding Is Linked to Dimerization of the Regulatory Domain of Phenylalanine Hydroxylase

Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer–dimer equilibrium with a dissociation constant of ∼46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (<i>K</i>_d = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme

The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (<i>S</i>)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains

Mechanistic Studies of an Amine Oxidase Derived from d‑Amino Acid Oxidase

The flavoprotein d-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant d-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) <i>Angew. Chem., Int. Ed. 53</i>, 4428–4431]. We describe here the use of pH and kinetic isotope effects with (<i>R</i>)-α-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the same catalytic mechanism as the wild-type enzyme. The effects of pH on the steady-state and rapid-reaction kinetics establish that the neutral amine is the substrate, while an active-site residue, likely Tyr224, must be uncharged for productive binding. There is no solvent isotope effect on the <i>k</i>_cat/<i>K</i>_m value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the <i>k</i>_cat/<i>K</i>_m value is pH-independent, with an average value of 5.3, similar to values found with amino acids as substrates for the wild-type enzyme and establishing that there is no commitment to catalysis with this substrate. The <i>k</i>_cat/<i>K</i>_O₂ value is similar to that seen with amino acids as the substrate, consistent with the oxidative half-reaction being unperturbed by the mutation and with flavin oxidation preceding product release. All of the data are consistent with the mutant enzyme utilizing the same mechanism as the wild-type enzyme, transfer of hydride from the neutral amine to the flavin

Isotope Effects Suggest a Stepwise Mechanism for Berberine Bridge Enzyme

The flavoprotein Berberine Bridge Enzyme (BBE) catalyzes the regioselective oxidative cyclization of (<i>S</i>)-reticuline to (<i>S</i>)-scoulerine in an alkaloid biosynthetic pathway. A series of solvent and substrate deuterium kinetic isotope effect studies were conducted to discriminate between a concerted mechanism, in which deprotonation of the substrate phenol occurs before or during the transfer of a hydride from the substrate to the flavin cofactor and substrate cyclization, and a stepwise mechanism, in which hydride transfer results in the formation of a methylene iminium ion intermediate that is subsequently cyclized. The substrate deuterium isotope effect of 3.5 on <i>k</i>_red, the rate constant for flavin reduction, is pH-independent, indicating that C–H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on <i>k</i>_red are equal to 1 for both wild-type BBE and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism as a possibility for catalysis by BBE. An intermediate was not detected by rapid chemical quench or continuous-flow mass spectrometry experiments, indicating that it must be short-lived

Mechanism of Flavoprotein l‑6-Hydroxynicotine Oxidase: pH and Solvent Isotope Effects and Identification of Key Active Site Residues

The flavoenzyme l-6-hydroxynicotine oxidase is a member of the monoamine oxidase family that catalyzes the oxidation of (<i>S</i>)-6-hydroxynicotine to 6-hydroxypseudooxynicotine during microbial catabolism of nicotine. While the enzyme has long been understood to catalyze oxidation of the carbon–carbon bond, it has recently been shown to catalyze oxidation of a carbon–nitrogen bond [Fitzpatrick, P. F., et al. (2016) <i>Biochemistry</i> <i>55</i>, 697–703]. The effects of pH and mutagenesis of active site residues have now been utilized to study the mechanism and roles of active site residues. Asn166 and Tyr311 bind the substrate, while Lys287 forms a water-mediated hydrogen bond with flavin N5. The N166A and Y311F mutations result in ∼30- and ∼4-fold decreases in <i>k</i>_cat/<i>K</i>_m and <i>k</i>_red for (<i>S</i>)-6-hydroxynicotine, respectively, with larger effects on the <i>k</i>_cat/<i>K</i>_m value for (<i>S</i>)-6-hydroxynornicotine. The K287M mutation results in ∼10-fold decreases in these parameters and a 6000-fold decrease in the <i>k</i>_cat/<i>K</i>_m value for oxygen. The shapes of the pH profiles are not altered by the N166A and Y311F mutations. There is no solvent isotope effect on the <i>k</i>_cat/<i>K</i>_m value for amines. The results are consistent with a model in which both the charged and neutral forms of the amine can bind, with the former rapidly losing a proton to a hydrogen bond network of water and amino acids in the active site prior to the transfer of hydride to the flavin

Structure of the Flavoprotein Tryptophan 2‑Monooxygenase, a Key Enzyme in the Formation of Galls in Plants

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to yield indole-3-acetamide. This is the initial step in the biosynthesis of the plant growth hormone indole-acetic acid by bacterial pathogens that cause crown gall and related diseases. The structure of the enzyme from <i>Pseudomonas savastanoi</i> has been determined by X-ray diffraction methods to a resolution of 1.95 Å. The overall structure of the protein shows that it has the same fold as members of the monoamine oxidase family of flavoproteins, with the greatest similarities to the l-amino acid oxidases. The location of bound indole-3-acetamide in the active site allows identification of residues responsible for substrate binding and specificity. Two residues in the enzyme are conserved in all members of the monoamine oxidase family, Lys365 and Trp466. The K365M mutation decreases the <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_Trp values by 60000- and 2 million-fold, respectively. The deuterium kinetic isotope effect increases to 3.2, consistent with carbon–hydrogen bond cleavage becoming rate-limiting in the mutant enzyme. The W466F mutation decreases the <i>k</i>_cat value <2-fold and the <i>k</i>_cat/<i>K</i>_Trp value only 5-fold, while the W466M mutation results in an enzyme lacking flavin and detectable activity. This is consistent with a role for Trp466 in maintaining the structure of the flavin-binding site in the more conserved FAD domain

HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase

Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an <i>S</i> = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (<i>A</i>_iso = 0.0 ± 0.3 MHz) and dipolar couplings (<i>T</i> = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an <i>A</i>_iso of 2.0 MHz and a <i>T</i> of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH^– for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an <i>A</i>_iso of 0.0 MHz and a dipolar coupling (<i>T</i> = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin

Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate Rearrangements in the Formation of the Oxygen-Reactive Complex

Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O₂ surrogate to poise Fe­(II) at the catalytic site in an <i>S</i> = ³/₂, {FeNO}⁷ form amenable to EPR spectroscopy. ²H-electron spin echo envelope modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}⁷ paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}⁷ center to the closest deuteron on 6,7-²H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-²H-tyrosine resulted in reorientation of the magnetic axes of the <i>S</i> = ³/₂, {FeNO}⁷ center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe­(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase