Showing papers by "Paul F. Fitzpatrick published in 2005"
TL;DR: nitroalkane oxidase (NAO) is the only flavoprotein to date for which a carbanion is definitively established as an intermediate in catalysis, and recent structural analyses show that NAO is homologous to the acyl-CoA dehydrogenase and acyl
45 citations
TL;DR: The present results are consistent with cyanide trapping this electrophilic intermediate and removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5).
Abstract: The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2-2H2-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for nitroalkane oxidase involves removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent w...
34 citations
TL;DR: The similarities of the isotope effects and extent of tunneling establish that the reactivities of the hydroxylating intermediates in the three enzymes are essentially identical.
Abstract: Deuterium kinetic isotope effects for hydroxylation of the methyl group of 4-methylphenylalanine have been used as a probe of the relative reactivities of the hydroxylating intermediates in the aromatic amino acid hydroxylases phenylalanine, tyrosine, and tryptophan hydroxylase. When there are three deuterium atoms in the methyl group, all three enzymes exhibit an intrinsic isotope effect of about 13. The temperature dependence of the isotope effect is consistent with moderate tunneling, with the extent of tunneling identical for all three enzymes. In the case of phenylalanine hydroxylase, the presence of the regulatory domain has no effect on the values. The intrinsic primary and secondary isotope effects were determined using 4-methylphenylalanine containing one or two deuterium atoms in the methyl group. With one deuterium atom, the intrinsic primary and secondary effects have average values of 10 and 1.1, respectively. With two deuterium atoms, the primary effects decrease to 7.4 and the secondary eff...
32 citations
TL;DR: The increased stability of S8E, S19E, and S31E tyrosine hydroxylases may be one of the physiological effects of phosphorylation, which may also have implications for the interpretation of activities of heterogeneous mixtures of tyrosines which have been phosphorylated.
27 citations
TL;DR: The kinetic properties of MTOX with the slow substrate sarcosine were determined and stopped-flow spectroscopy with both the protiated and deuterated substrate failed to detect any intermediates between the enzyme-substrate complex and the fully reduced enzyme.
Abstract: N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of secondary amino acids such as N-methyltryptophan or N-methylglycine (sarcosine). MTOX is one of several flavin-dependent amine oxidases whose chemical mechanism is still debated. The kinetic properties of MTOX with the slow substrate sarcosine were determined. Initial rate data are well-described by the equation for a ping-pong kinetic mechanism, in that the V/K(O)()2 value is independent of the sarcosine concentration at all accessible concentrations of oxygen. The k(cat)/K(sarc) pH profile is bell-shaped, with pK(a) values of 8.8 and about 10; the latter value matches the pK(a) value of the substrate nitrogen. The k(cat) pH profile exhibits a single pK(a) value of 9.1 for a group that must be unprotonated for catalysis. There is no significant solvent isotope effect on the k(cat)/K(sarc) value. With N-methyl-(2)H(3)-glycine as the substrate, there is a pH-independent kinetic isotope effect on k(cat), k(cat)/K(sarc), and the rate constant for flavin reduction, with an average value of 7.2. Stopped-flow spectroscopy with both the protiated and deuterated substrate failed to detect any intermediates between the enzyme-substrate complex and the fully reduced enzyme. These results are used to evaluate proposed chemical mechanisms.
22 citations
TL;DR: The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate, finding that at pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k(cat) value for turnover, establishing reduction as rate-limiting.
Abstract: In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and Nl-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with Nl,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k c a t value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k c a t value decreases below a pK a of 9.0. The k c a t /K m value for BESPM exhibits a bell-shaped pH profile, with pK a values of 9.8 and 10.8. These pK a values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k c a t value and the k c a t /K m value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.
22 citations