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A study examines the structural properties of the active site cavities in human and rat monoamine oxidases (MAOA and MAOB) in their detergent-purified and outer mitochondrial membrane (OMM) bound forms. The data shows that the structural properties of MAOA and MAOB active sites are distinctly different in human, whereas in rat they are similar.

Classical monoamine oxidases and copper-containing semicarbazide-sensitive amine oxidases are drug targets for a variety of established and novel pharmaceuticals used to treat conditions such as depression, Parkinsonism and inflammatory disorders. Development of enzyme inhibitors, both historically and currently, assumes an underlying adherence of these enzymes to Michaelis-Menten kinetic principles. In this mini-review, we discuss historical data from several laboratories and novel data from our own laboratories which show clearly that such an assumption is invalid. Rather, these enzymes often display hormetic behaviour towards their substrates, resulting in bell-shaped kinetic plots. We outline possible underlying mechanisms which might account for this behaviour and show how novel reversible hormetic drugs may capitalise on these mechanisms to introduce a new dimension in selectivity. The potential future benefits for therapeutic modulation of amine oxidase activities are discussed. Key words: enzyme kinetics, amine oxidases, substrates, imidazolines, cations, INTRODUCTION Mammalian amine oxidase enzymes play critically important roles in physiological and pathological tissue development and function. These roles are, for the most part, directly related to the enzymatic capacity [...]

Tyr435 in human monoamine oxidase B (MAO B) was mutated with the amino acids Phe, His, Leu, or Trp, the mutant proteins expressed in Pichia pastoris, and purified to homogeneity to probe the significance of the structure. The results provide new insights into the role of these aromatic residues in the catalytic reductive phase of amine oxidation.

The three-dimensional structure of recombinant human monoamine oxidase A (hMAO A) as its clorgyline-inhibited adduct is described. Although the chain-fold of hMAO A is similar to that of rat MAO A and human MAO B (hMAO B), hMAO A is unique in that it crystallizes as a monomer and exhibits the solution hydrodynamic behavior of a monomeric form rather than the dimeric form of hMAO B and rat MAO A. hMAO A's active site consists of a single hydrophobic cavity of [approximately or equal to] 550 [[Angstrom].sup.3], which is smaller than that determined from the structure of deprenyl-inhibited hMAO B ([approximately equal to] 700 [[Angstrom].sup.3]) but larger than that of rat MAO A ([approximately equal to] 450 [[Angstrom].sup.3]). An important component of the active site structure of hMAO A is the loop conformation of residues 210-216, which differs from that of hMAO B and rat MAO A. The origin of this structural alteration is suggested to result from long-range interactions in the monomeric form of the enzyme. In addition to serving as a basis for the development of hMAO A specific inhibitors, these data support the proposal that hMAO A involves a change from the dimeric to the monomeric form through a Glu-151 [right arrow] Lys mutation that is specific of hMAO A [Andres, A. M., Soldevila, M., Navarro, A., Kidd, K. K., Oliva, B. & Bertranpetit, J. (2004) Hum. Genet. 115, 377-386]. These considerations put into question the use of MAO A from nonhuman sources in drug development for use in humans. flavin | neurotransmitter | membrane protein | antidepressant target

Research has been conducted on the monoamine oxidase A. The role of the tyrosyl residues which are located near the covalent 8(alpha)-S-cysteinyl FAD in this oxidase has been investigated and the results are reported.

The activation of dioxygen at the carboxylate-bridged diiron (II) cluster in the R2 subunit of Escherichia coli class I ribonucleotide reductase produces the enzyme's catalytically essential stable tyrosyl radical by one-electron oxidation of tyrosine 122. Evidence for the trysptophan cation radical assignment is provided by showing that the abdorbing species reacts rapidly with reductants.

The mechanism and outcome of dioxygen activation by the carboxylate-bridged diiron(II) cluster in the W48F site-directed variant of protein R2 of ribonucleotide reductase from Escherichia coli are determined by kinetic, spectroscopic and chemical methods. The result provides convincing evidence for the rapid and nearly quantitative formation of the Fe(IV)Fe(III) cluster and the Y122 radical species as a consequence of the defect in electron injection in the W48F-substituted variant.

The binding of phenethylamine analogues to monoamine oxidase A depends on steric interactions between the alkyl side chains and the enzyme. The binding site for the aryl ring of these inhibitors is the same regardless of which analogue is used.

Tetrahydrobiopterin (H(sub)4B) participates in electron transfer during the production of nitric oxide by nitric oxide synthase. Researchers used EPR to identify a one-electron oxidized form of H(sub)4B.

An analysis of the mechanism of ferroxidase reaction of ferritin shows that there exists a diferric mu-1,2 bridging transient peroxide intermediate similar to other O2-activating non-heme diiron proteins. This was evident in an experiment that characterized the intermediate and established the peroxodiferric assignment using the rapid freeze-quenching technique. Discrete vibrational modes were detected in the ferritin, suggesting that a single chromophore exists in a homogeneous state.

The ferroxide reaction catalyzed by recombinant frog M ferritin under low-iron loading was examined using rapid freeze-quench Mossbauer and stopped-flow absorption spectroscopy. Oxidation of Fe2+ ions with Mossbauer parameters resulted to the formation of a single transient iron species indicative of a peroxidiferric compound. The similarity of the reaction from those of the reaction of O2 with MMOH and with DE84E-R2 mutant suggest a similar mechanism for ferritin ferroxidation and for oxygen activation reaction by diiron proteins.

Freeze-quench electron paramagnetic resonance studies were carried out in the investigation of the enzyme kinetics of quinol reductase cytochrome bo3 from Escherichia coli under conditions of substrate limitation. This method allowed the researcher to accurately measure the potential of the heme b prosthesis and relate this to the electron transfer mechanics. Results indicate that proton translocation takes place by a Q-cycle mechanism.

Research was conducted to investigate the overproduction of the Azospirillum brasilense glutamate synthase alpha subunit in Escherichia coli. Standard protocols were utilized to carry out DNA manipulations while a T7 sequencing kit was used for DNA sequencing. Results showed that the absorption spectrum of the iron-sulfur flavoprotein characterized the purified alpha subunit of glutamate synthase. Moreover, evidence was obtained concerning changes that took place with the alpha subunit on the binding of the pyridine nucleotide to the beta subunit.

The mechanism of ferritin Fe uptake was studied to characterize the type of Fe3+ intermediates that form after ferroxidation and during the initial process of mineralization. The Mossbauer spectroscopic investigation shows that several multinuclear (n=2-3)Fe3+ - oxy clusters form in parallel with the rapid Fe2+ oxidation. This suggests that the final formation of the ferritin mineral requires transporting and multiple processes.

A reanalysis of Mossbauer spectroscopy using results from earlier studies on the paramagnetic intermediate, X, in the cofactor assembly reaction in Escherichia coli ribonucleotide reductase was conducted for a fuller description of its electronic structure. Results showed isomer shifts of delta(FeA) = 0.56(3) mm/s and delta(Feb) = 0.26(4) mm/s. According to the reduced isomer shift and the hyperfine anisotropy of FeB, X can be best described as a spin-coupled Fe(super III)/Fe(super IV) center without a radical, but with significant spin delocalization onto the oxygen ligand(s).

Xanthine dehydrogenase (XDH) is a metalloflavoprotein that catalyzes the hydroxylation of xanthine. Much of the research on XDH structure and function has come from preparations sourced from eukaryotes such as chicken or rat liver XDH. To further explore the catalytic, structural and cofactor properties of the enzyme, prokaryotic XDH was obtained from Comamonas acidovorans. Results show that prokaryotic XDH has properties similar to those of the better-known eukaryotic enzymes.

The interactions among the soluble methane monooxygenase (sMMO) enzyme system from Methylococcus capsulatus (Bath) constituents, protein B, reductase, and hydroxylase is analyzed by observing the conversion of nitrobenzene to nitrophenol via single turnover and catalytic conditions. Single turnover reactions between Hred and nitrobenzene depend on the concentration of protein B in the system. The effects of protein B components and reductase on the kinetic constants and the reaction of Hred with nitrobenzene and dioxygen are discussed.

Kinetic studies of the hydrogen tunneling phenomenon in bovine liver monoamine oxidase B indicate the mechanism to be a single-step hydrogen abstraction involving a late transition state. Isotopic effects and their exponents show that many rate-limiting steps are constant across low temperature ranges, while the isotopic effects are more at low pH values. These studies also give values for the Arrhenius factors.

Mossbauer and EPR spectroscopic studies of the intermediate X associated with the assembly of tyrosyl radical-diiron(III) (.Y122) cofactor of Escherichia coli ribonucleotide reductase reveal that X is a spin coupled system that contains two high-spin ferric ions and a free radical. Spontaneous cofactor assembly reaction with Fe(2+) and O2 suggests that intermediates produced from Y122 does not contain Fe(IV). Spectroscopic data yields information on the similarities between the radical component in the diferric radical precursor and the R2 cofactor.

Stopped-flow absorption, rapid freeze-quench EPR and rapid freeze-quench Mossbauer spectroscopic studies help examine kinetics of assembly of the tyrosyl radical-diiron(III) cofactor of Escherichia coli ribonucleotide reductase with limiting Fe(2+). Kinetic data and amount of diferric radical species .Y122 suggest that a 560 nm absorbing transient species produces .Y122. The 560 nm absorbing species is a tryptophan radical cation generated by oxidation of W48. Kinetic studies support the noninvolvement of Fe(IV) intermediates in the generation of .Y122.

Stopped-flow absorption, rapid freeze-quench EPR and rapid freeze-quench Mossbauer spectroscopic studies of the kinetics of assembly of the tyrosyl radical-diiron(III) cofactor of Escherichia coli ribonucleotide reductase in the presence of excess Fe(2+) reveal that the mechanism involves two sequential first-order reactions the first of which involves accumulation of diferric radical species. The second reaction involves degradation producing . Y122 and the oxo-bridged diferric cluster. The intermediate X oxidizes Y122 to the cofactor and Fe(III) containing species.

Anaerobic titrations of reduced cytochrome c3 with flavodoxin reveal a stoichiometry of 4 moles of flavodoxin in the oxidization of the tetraheme cytochrome. Anaerobic stopped flow methods help study the kinetic properties of electron transfer. Electron transfer occurs between reduced Desulfovibrio vulgaris cytochrome c3 and Desulfovibrio vulgaris flavodoxins. The oxidization reaction involving electron transfer produces only neutral flavodoxin semiquinone and oxidized cytochrome c3.

Steady-state and reductive half-reaction anaerobic stopped-flow kinetics techniques are used to analyze the influence of para and meta substituents on the bovine liver mitochondrial monoamine oxidase B (MAO B) catalyzed oxidative deamination reaction. The catalytic mechanism is not changed in different substitution reactions but different interactions are observed. Hydrophobicity of the substituents influences the binding capacity to MAO B.