Showing papers by "Paul F. Fitzpatrick published in 2007"
TL;DR: Rapid-freeze quench Mossbauer spectroscopy has now provided direct evidence for the presence of an Fe(IV) intermediate in the reaction catalyzed by tyrosine hydroxylase.
Abstract: Tyrosine hydroxylase, a member of the aromatic amino acid hydroxylase family, uses a mononuclear Fe(II) and tetrahydropterin for hydroxylation of tyrosine to dihydroxyphenylalanine. Rapid-freeze quench Mossbauer spectroscopy has now provided direct evidence for the presence of an Fe(IV) intermediate in the reaction catalyzed by tyrosine hydroxylase. Rapid-quench techniques provide support for the kinetic competence of this species as the hydroxylating intermediate. This is the first direct evidence for a mononuclear Fe(IV) intermediate in an enzymatic aromatic hydroxylation reaction.
161 citations
TL;DR: The mechanism of N-methyltryptophan oxidase, a flavin-dependent amine oxidase from Escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations to distinguish between some discrete electron-transfer mechanisms and a direct hydride-transfer mechanism.
Abstract: The mechanism of N-methyltryptophan oxidase, a flavin-dependent amine oxidase from Escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations The 15(kcat/Km) kinetic isotope effect for sarcosine oxidation is pH-dependent with a limiting value of 0994-0995 at high pH Density functional theory calculations on model systems were used to interpret these isotope effects The isotope effects are inconsistent with proposed mechanisms involving covalent amine-flavin adducts but cannot by themselves conclusively distinguish between some discrete electron-transfer mechanisms and a direct hydride-transfer mechanism, although the latter mechanism is more consistent with the energetics of the reaction
74 citations
TL;DR: Deuterium and solvent isotope effects with wild-type and mutant variants of the lactate dehydrogenase flavocytochrome b(2) show that OH and CH bond cleavage are not concerted, but become so in the Y254F enzyme, consistent with a highly asynchronous reaction in which OH bond cleaving precedes hydride transfer.
Abstract: Deuterium, solvent, and (15)N kinetic isotope effects have been used to probe the mechanisms by which flavoproteins oxidize carbon-oxygen and carbon-nitrogen bonds in amines, hydroxy acids, and alcohols. For the amine oxidases d-amino acid oxidase, N-methyltryptophan oxidase, and tryptophan monooxygenase, d-serine, sarcosine, and alanine are slow substrates for which CH bond cleavage is fully rate limiting. Inverse isotope effects for each of 0.992-0.996 are consistent with a common mechanism involving hydride transfer from the uncharged amine. Computational analyses of possible mechanisms support this conclusion. Deuterium and solvent isotope effects with wild-type and mutant variants of the lactate dehydrogenase flavocytochrome b(2) show that OH and CH bond cleavage are not concerted, but become so in the Y254F enzyme. This is consistent with a highly asynchronous reaction in which OH bond cleavage precedes hydride transfer. The results of Hammett analyses and solvent and deuterium isotope effects support a similar mechanism for alcohol oxidase.
22 citations
TL;DR: His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton, and the effects of mutating this residue to glutamine have been determined to provide further insight into its role.
Abstract: His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q ...
20 citations
TL;DR: All the syntheses were carried out by simple protection and deprotection steps from commonly used selective protecting reagents and these deuterium labeled compounds can be used as mechanistic probes of polyamine oxidizing enzymes.
Abstract: The synthesis of deuterium labeled spermine, N(1)-acetylspermine and N(1)-acetylspermidine is reported. 1,1,3,3-(2)H(4)-N(1)-Acetylspermine hydrochloride, 1,1,3,3-(2)H(4)-N(1)-acetylspermidine hydrochloride and 1,1,3,3,10,10,12,12-(2)H(8)-spermine dihydrochloride were obtained in seven, four and three steps respectively. All the syntheses were carried out by simple protection and deprotection steps from commonly used selective protecting reagents. These deuterium labeled compounds can be used as mechanistic probes of polyamine oxidizing enzymes.
5 citations