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P F Fitzpatrick

Publications and source records attributed to P F Fitzpatrick.

At least 55 records · Page 3Linked to original sources
PubMed1995

Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 1. Kinetic mechanism.

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indole-3-acetamide, carbon dioxide, and water. The kinetic mechanism of the enzyme has been determined with tryptophan as substrate at pH 8.3. Initial velocity patterns, when both amino acid and oxygen concentrations are varied, are sequential with tryptophan and ping-pong with phenylalanine and methionine. Reduction by tryptophan in the absence of oxygen is biphasic. The rate of the rapid phase varies with the tryptophan concentration, with a limiting rate of 139 s-1 and an apparent Kd value of 0.11 mM. There is a primary deuterium kinetic isotope effect on the limiting rate of reduction of 2.4. The rapid phase is followed by a slow, concentration and isotope-independent phase that is much slower than turnover; this is ascribed to dissociation of a reduced enzyme-imino acid complex. In the absence of oxygen, tryptophan is converted to indolepyruvate imine. The rate of this reaction is the same as that of the rapid phase in the reduction. Reaction of the reduced enzyme-imino acid complex with oxygen to form oxidized flavin is monophasic, with a rate constant of 196 mM-1 s-1; no intermediates are detectable. The rate of formation of indole-3-acetamide agrees with the rate of reaction with oxygen. This is followed by slow product dissociation.

Hydrogen-Ion Concentration↗
PubMed1995

Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 2. pH and kinetic isotope effects.

pH and kinetic isotope effects on steady-state kinetic parameters have been determined for the flavoprotein tryptophan 2-monooxygenase with tryptophan, phenylalanine, 2-hydrazino-3-propanoic acid, and methionine as substrates. The V/K values of the amino acid substrates show that a residue with an apparent pKa value of 5 must be unprotonated for activity, a residue with a pKa value equal to that of the amino group of the substrate must be protonated, and deprotonation of a residue with pKa value of 10 increases the V/K value. A group in the free enzyme with a pKa value of 6 must be deprotonated for tight binding of amide inhibitors and protonated for tight binding of acids, establishing this as the intrinsic pKa value. The temperature dependence of this pKa value is consistent with involvement of a histidinyl residue. Deprotonation of the residue with a pKa value of 10 decreases binding of amide inhibitors. The D(V/Ktrp) value is less than 1.7 between pH 5 and 10, consistent with a forward commitment to catalysis of 7-15 with this substrate. The D(V/K)met value is pH dependent, increasing from a minimal value of 1.8 at pH 8.3 to a limiting value of 5.3 at both high and low pH, with pKa values of 5.1 and 10. The increase in both the isotope effect and the V/Kmet value at high pH is consistent with a conformational change to a more open active site above pH 10. The D(V/K)ala value is 5.3 at pH 8.3; this is probably the intrinsic isotope effect with this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen-Ion Concentration↗
PubMed1995

Purification and characterization of the flavoprotein tryptophan 2-monooxygenase expressed at high levels in Escherichia coli.

Tryptophan 2-monooxygenase from Pseudomonas savastanoi is a flavoprotein which catalyzes the formation of indoleacetamide from tryptophan. This is the first step in a two-step pathway for the formation of indoleacetic acid during infection of plants and subsequent gall formation by this and other bacteria. The enzyme has been expressed in Escherichia coli at high levels, and a purification procedure has been developed which generates micromolar amounts of protein. The purified enzyme contains tightly bound indoleacetamide; a method involving dialysis against 20% methanol has been developed for removing the indoleacetamide without significant loss of enzyme activity. Amino acids with large hydrophobic side chains are the best substrates; N-substituted phenylalanines will also act as substrates. N-ethylmaleimide, methyl methanethiol-sulfonate, and diethylpyrocarbonate act as active site-directed reagents, consistent with a histidine and a cysteine at or near the enzyme active site. Vinylglycine partially inactivates the enzyme, while propargylglycine has no effect.

Amino Acids↗
PubMed1995

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.

Amino Acid Sequence↗
PubMed1994

pH and kinetic isotope effects on the oxidative half-reaction of D-amino-acid oxidase.

D-Amino-acid oxidase catalyzes the oxidation of D-amino acids to imino acids. In the oxidative half-reaction, oxygen reacts with the reduced enzyme-imino acid complex to reoxidize the bound FAD. This is then followed by dissociation of the imino acid. The effects of pH and D2O on the kinetics of the oxidative half-reaction of D-amino-acid oxidase have been determined with glycine, D-alanine, and D-serine as substrates. Reaction of the reduced enzyme with oxygen requires that a group with a pKa value of about 10.5 be protonated and a group with a pKa value of 8.5 be deprotonated. The former value is not seen with D-alanine as substrate; the latter is only seen with glycine. No solvent isotope effects are seen on the V/KO2 value with D-alanine, consistent with rate-limiting electron transfer. Product release involves a pH-dependent conformational change. This is rate-limiting at all pH values with D-alanine as substrate. Significant solvent isotope effects are seen on the Vmax value with D-alanine. The proton inventory at high pH is linear, consistent with release of a single proton in the slow step; at pH 6 the solvent inventory is bowl-shaped, consistent with a solvent isotope effect on the conformation of the protein. With glycine the DV value increases to the intrinsic value at pH 10.5; this establishes that CH bond cleavage becomes rate-limiting with this substrate above pH 10.

Alanine↗
PubMed1994

Bifunctional peptidylglcine alpha-amidating enzyme requires two copper atoms for maximum activity.

The conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides occurs in two distinct reactions, both of which are catalyzed by bifunctional peptidylglycine alpha-amidating enzyme. The first step is the alpha-hydroxylation of the C-terminal glycine residue and the second step is the dealkylation of the alpha-hydroxyglycine-extended peptide to the alpha-amidated peptide and glyoxylate. We show that the bifunctional enzyme requires 1.9 +/- 0.2 mol of copper/mol of enzyme for maximal dansyl-Tyr-Lys-Gly amidation activity under the conditions of high enzyme concentration (approximately 80 microM) required to measure initial rates for this poor substrate. The enzyme, as purified, contains a substoichiometric amount of copper and has only trace levels of amidation activity. Addition of exogenous Cu(II) ions stimulates amidation activity approximately 3000-fold at the optimum copper stoichiometry and the enzyme is then inhibited by excess Cu(II). No stimulation of amidation activity is observed upon the addition of the following divalent metal ions: Mn(II), Fe(II), Ni(II), Cd(II), and the oxovanadium cation, VO(II). The enzyme-catalyzed dealkylation of alpha-hydroxyhippuric acid to benzamide shows no dependence on copper, indicating that the copper dependence of the amidation reaction must be attributed to a copper dependence in peptide alpha-hydroxylation.

Amino Acid Sequence↗
PubMed1994

Intrinsic primary, secondary, and solvent kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase: evidence against a concerted mechanism.

D-Amino acid oxidase catalyzes the oxidation of D-amino acids to imino acids with subsequent transfer of the electrons to molecular oxygen. Proposed mechanisms for the mode of cleavage of the substrate CH bond include stepwise formation of a carbanion, followed by attack of the carbanion on the enzyme-bound FAD, direct hydride transfer of the substrate alpha-hydrogen to the FAD, and transfer of a hydride from the substrate amino group to the FAD. Conditions have previously been established under which large, limiting, primary deuterium kinetic isotope effects can be measured with D-alanine, D-serine, and glycine as substrates for D-amino acid oxidase [Denu, J. M., & Fitzpatrick, P. F. (1992) Biochemistry 31, 8207-8215]. To determine whether these values are the intrinsic isotope effects, primary tritium kinetic isotope effects have been determined with these three substrates. The values are 12.6, 8.6, and 6.4, respectively. These values are consistent with expression of the intrinsic isotope effects under these conditions, allowing for determination of the values of the intrinsic deuterium effects as 5.7, 4.5, and 3.6 for D-alanine, D-serine, and glycine, respectively. Under these conditions, the alpha-secondary tritium kinetic isotope effect with glycine, the beta-secondary deuterium kinetic isotope effect with D-alanine, and the solvent kinetic isotope effect with D-serine are all indistinguishable from unity. These results are not consistent with concerted mechanisms for CH bond cleavage with this enzyme, but are fully consistent with the involvement of a carbanion intermediate.

Alanine↗
PubMed1993

Identification of the intersubunit binding region in rat tyrosine hydroxylase.

Limited proteolysis converts the 39200 molecular weight catalytic domain of rat tyrosine hydroxylase to a monomer with a molecular weight of 37600. The purified monomer is almost fully active, with minor changes in kinetic parameters at pH 7. Mass spectral analysis and N-terminal sequencing of the proteolytically generated species establish that 20 amino acids have been removed from the carboxyl terminus and five from the amino terminus. Based on these results, the carboxyl terminus is responsible for tetramer formation by tyrosine hydroxylase. The sequence of amino acids which is removed is consistent with a coiled coil structure in the intact tetramer.

Amino Acid Sequence↗
PubMed1993

Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.

Amino Acid Sequence↗
PubMed1993

Lysine241 of tyrosine hydroxylase is not required for binding of tetrahydrobiopterin substrate.

The lysine residues at positions 194 and 198 in phenylalanine hydroxylase have been shown to react with a photoaffinity label which is an analog of phenyltetrahydropterin (Gibbs, B. S., and Benkovic, S. J. (1991) Biochemistry 30, 6795-6802), in a manner suggesting that these lysine residues are involved in tetrahydrobiopterin binding. The related enzyme tyrosine hydroxylase has a lysine at position 241 which, given the 75% identity between its C-terminal 330 amino acids and those of phenylalanine hydroxylase, corresponds to lysine194 of phenylalanine hydroxylase. Site-directed mutagenesis was used to alter lysine241 of tyrosine hydroxylase to alanine. Steady-state kinetic parameters were measured for wild-type and K241A tyrosine hydroxylase. No kinetic parameter differed between the wild-type and K241A enzymes, including Vmax values, Michaelis constants for tetrahydrobiopterin, 6-methyl-tetrahydropterin, and tyrosine, and the inhibition constants for norepinephrine. These results show that lysine241 is not required for tetrahydrobiopterin binding to tyrosine hydroxylase.

Amino Acid Sequence↗
PubMed1992

pH and kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase.

Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pKa values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration (approximately 15 microM) at pH 9,D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKa values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of approximately 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H3O+ and H2O below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kgly value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.

Alanine↗
PubMed1992

Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity.

Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P. F., Chlumsky, L. J., Daubner, S. C., and O'Malley, K. L. (1990) J. Biol. Chem. 265, 2042-2047). The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of [32P]phosphate. Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7. Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate. Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7. Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold. Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold. The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells. In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions. These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines.

Amino Acid Sequence↗
PubMed1992

The amino acid substrate of bovine tyrosine hydroxylase.

Tyrosine hydroxylase catalyzes the tetrahydropterin-dependent hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine. Several nonphysiological aromatic amino acids have been examined as inhibitors and substrates for bovine adrenal tyrosine hydroxylase. The Ki values for para-substituted phenylalanines increase as the size of the substituent increases. For each A2 increase in surface area of the substituent, the free energy of binding becomes 50 cal more positive. Replacement of the phenyl ring with a pyridyl ring decreases the affinity about one order of magnitude. A number of these aromatic amino acids are also substrates for the enzyme. The KM values again increase in size with increasing size of the substituent, but the Vmax value is independent of the reactivity of the amino acid. The effect of size on binding is consistent with a tight interaction between the para position region of the substrate and the enzyme. The lack of a change in the Vmax value is consistent with the rate-limiting step in catalysis by bovine tyrosine hydroxylase being formation of the hydroxylating intermediate rather than hydroxylation of the amino acid. These results will be useful in designing mechanism-based inhibitors of catecholamine biosynthesis and establish that the mechanisms of rat and bovine tyrosine hydroxylase do not differ significantly.

Adrenal Glands↗
PubMed1991

Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogues.

Tyrosine hydroxylase catalyzes the formation of dihydroxyphenylalanine from tyrosine, utilizing a tetrahydropterin and molecular oxygen as cosubstrates. Several approaches were taken to examining the identity of the rate-limiting step in catalysis. Steady-state kinetic parameters were determined with a series of ring-substituted phenylalanines. The Vmax value was unchanged with substrates ranging in reactivity from tyrosine to 4-fluorophenylalanine. Neither 4-pyridylalanine N-oxide, a model of tyrosine phenoxide, nor 4-hydroxy-3-pyridylalanine N-oxide or alpha-amino-3-hydroxy-4-pyridone-1- propionic acid, models of a hydroxycyclohexadienone intermediate, was an effective inhibitor. There was no solvent isotope effect on either the Vmax or the V/KTyr value. These results establish that no chemistry occurs at the amino acid in the rate-limiting step and no exchangeable proton is in flight in the rate-limiting step. The results are consistent with a model in which the slow step in catalysis is formation of the hydroxylating intermediate.

Adrenal Glands↗
PubMed1991

Steady-state kinetic mechanism of rat tyrosine hydroxylase.

The steady-state kinetic mechanism for rat tyrosine hydroxylase has been determined by using recombinant enzyme expressed in insect tissue culture cells. Variation of any two of the three substrates, tyrosine, 6-methyltetrahydropterin, and oxygen, together at nonsaturating concentrations of the third gives a pattern of intersecting lines in a double-reciprocal plot. Varying tyrosine and oxygen together results in a rapid equilibrium pattern, while the other substrate pairs both fit a sequential mechanism. When tyrosine and 6-methyltetrahydropterin are varied at a fixed ratio at different oxygen concentrations, the intercept replot is linear and the slope replot is nonlinear with a zero intercept, consistent with rapid equilibrium binding of oxygen. All the replots when oxygen is varied in a fixed ratio with either tyrosine or 6-methyltetrahydropterin are nonlinear with finite intercepts. 6-Methyl-7,8-dihydropterin and norepinephrine are competitive inhibitors versus 6-methyltetrahydropterin and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine, a competitive inhibitor versus tyrosine, shows uncompetitive inhibition versus 6-methyltetrahydropterin. At high concentrations, tyrosine is a competitive inhibitor versus 6-methyltetrahydropterin. These results are consistent with an ordered kinetic mechanism with the order of binding being 6-methyltetrahydropterin, oxygen, and tyrosine and with formation of a dead-end enzyme-tyrosine complex. There is no significant primary kinetic isotope effect on the V/K values or on the Vmax value with [3,5-2H2]tyrosine as substrate. No burst of dihydroxyphenylalanine production is seen during the first turnover. These results rule out product release and carbon-hydrogen bond cleavage as rate-limiting steps.

Animals↗
PubMed1990

Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.

Rat tyrosine hydroxylase has been expressed at high levels in Spodoptera frugiperda cells using a baculovirus expression system. A cDNA containing the coding region for PC12 tyrosine hydroxylase was inserted into the unique EcoRI site of the transfer vector pLJC8 to yield the recombinant vector pLJC9. Spodoptera frugiperda cells were then co-infected with pLJC9 and wild type Autographa californica nuclear polyhedrosis virus. Recombinant virus particles containing the cDNA for tyrosine hydroxylase were selected by hybridization with authentic tyrosine hydroxylase cDNA. Three recombinant viruses were plaque-purified. All expressed a protein of Mr = 55,000 which reacted with antibodies to tyrosine hydroxylase. Forty-eight h after infection of cells with recombinant virus, the specific activity of tyrosine hydroxylase in the cell lysate was 30-100 nmol of dihydroxyphenylalanine produced/min/mg, consistent with 5-10% of the cell protein being tyrosine hydroxylase. Purification from 2.1 g of cells gave 5.8 mg of enzyme with a specific activity of 1.7 mumol of dihydroxyphenylalanine/min/mg. The purified enzyme is a tetramer of identical subunits, containing one covalently bound phosphoryl residue and 0.1 iron atom/subunit. No carbohydrate was detectable. Steady state kinetic results with tetrahydrobiopterin as substrate are consistent with a sequential mechanism for binding of tyrosine and tetrahydrobiopterin. Substrate inhibition occurs at tyrosine concentrations above 50 microM. Steady state kinetic parameters at pH 6.5 are Vmax = 74 min-1, KBH4 = 21 microM, KTyr = 9.4 microM, and Ko2 less than or equal to 6 microM. The Vmax value shows a broad pH optimum around pH 7. The KBH4 value is pH-dependent, increasing from about 20 microM below pH 7 to about 100 microM above pH 8. The KTyr value is independent of pH between pH 6 and pH 8.5.

Animals↗