The kinetics of ammonia release during the catalytic cycle of pig plasma amine oxidase.
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ABSTRACT:
SUBMITTER: Rius FX
PROVIDER: S-EPMC1153694 | biostudies-other | 1984 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity.
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Project description:Electron nuclear double-resonance ('ENDOR') spectroscopic studies on pig plasma amine oxidase have been carried out at 15 K. Deuterium-exchange studies show the presence of two sets of exchangeable protons, probably from two water molecules; from the magnitude of their hyperfine couplings, one is assigned to be equatorially, and the other axially, co-ordinated. Only one 14N hyperfine coupling is observed, suggesting that the bonding of all amino acid (histidine) or organic cofactor ligands is similar. Upon addition of azide, a further hyperfine coupling to nitrogen is observed which is smaller than that observed for the native enzyme; the hyperfine couplings to the remaining nitrogens are slightly altered.
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Project description:Initial-velocity and product-inhibition studies on the enzyme benzylamine oxidase from pig plasma indicate that the order of substrate addition and product release is benzylamine on, ammonia off, oxygen on, hydrogen peroxide off, benzaldehyde off. Ammonia, but not benzaldehyde, is released under strictly anaerobic conditions which provides independent evidence for this order. Benzyl alcohol is a substrate for the enzyme. A chemical mechanism consistent with all the data is proposed.
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Project description:Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. gamma-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.
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Project description:Cytochrome c oxidase, the terminal protein in the respiratory chain, converts oxygen into water and helps generate the electrochemical gradient used in the synthesis of ATP. The catalytic action of cytochrome c oxidase involves electron transfer, proton transfer, and O2 reduction. These events trigger specific molecular changes at the active site, which, in turn, influence changes throughout the protein, including alterations of amino acid side chain orientations, hydrogen bond patterns, and protonation states. We have used IR difference spectroscopy to investigate such modulations for the functional intermediate states E, R2,Pm, and F. These spectra reveal deprotonation of its key glutamic acid E286 in the E and in the Pm states. The consecutive deprotonation and reprotonation of E286 twice within one catalytic turnover illustrates the role of this residue as a proton shuttle. In addition, the spectra point toward deprotonation of a redox-active tyrosine, plausibly Y288, in the F intermediate. Structural insights into the molecular mechanism of catalysis based on the subtle molecular changes observed with IR difference spectroscopy are discussed.
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Project description:The flavoenzyme monoamine oxidase (MAO) regulates mammalian behavioral patterns by modulating neurotransmitters such as adrenaline and serotonin. The mechanistic basis which underpins this enzyme is far from agreed upon. Reported herein is that the combination of a synthetic flavin and alloxan generates a catalyst system which facilitates biomimetic amine oxidation. Mechanistic and electron paramagnetic (EPR) spectroscopic data supports the conclusion that the reaction proceeds through a radical manifold. This data provides the first example of a biorelevant synthetic model for monoamine oxidase B activity.
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Project description:The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQamr) that is in equilibrium with a semiquinone radical (TPQsq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase from Arthrobacter globiformis. Anerobic soaking of the crystals with an amine substrate shifted the equilibrium toward TPQsq in an "on-copper" conformation, in which the 4-OH group ligated axially to the copper center, which was probably reduced to Cu(I). When the crystals were soaked with substrate in the presence of halide ions, which act as uncompetitive and noncompetitive inhibitors with respect to the amine substrate and dioxygen, respectively, the equilibrium in the crystals shifted toward the "off-copper" conformation of TPQamr. The halide ion was bound to the axial position of the copper center, thereby preventing TPQamr from adopting the on-copper conformation. Furthermore, transient kinetic analyses in the presence of viscogen (glycerol) revealed that only the rate constant in the step of TPQamr/TPQsq interconversion is markedly affected by the viscogen, which probably perturbs the conformational change. These findings unequivocally demonstrate that TPQ undergoes large conformational changes during the reductive half-reaction.
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Project description:The reaction between lentil (Lens culinaris) seedling amine oxidase and its chromogenic substrate, p-dimethylaminomethylbenzylamine, has been studied by the stopped-flow technique. Upon being mixed with substrate in the absence of oxygen, the enzyme is bleached in a complex kinetic process. A yellow intermediate absorbing at 464 nm and the first product (aldehyde) are formed in subsequent steps. When oxygenated buffer is mixed with substrate-reduced amine oxidase, the 496 nm absorption of the oxidized enzyme is very rapidly restored in a second-order process (k = 2.5 X 10(7) M-1 X S-1). This reaction is appreciable even at very low oxygen concentration, in keeping with the fairly low Km for O2 measured by steady-state kinetics.
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Project description:Although ADP release is the rate limiting step in product turnover by protein kinase A, the steps and motions involved in this process are not well resolved. Here we report the apo and ADP bound structures of the myristylated catalytic subunit of PKA at 2.9 and 3.5 Å resolution, respectively. The ADP bound structure adopts a conformation that does not conform to the previously characterized open, closed, or intermediate states. In the ADP bound structure, the C-terminal tail and Gly-rich loop are more closed than in the open state adopted in the apo structure but are also much more open than the intermediate or closed conformations. Furthermore, ADP binds at the active site with only one magnesium ion, termed Mg2 from previous structures. These structures thus support a model where ADP release proceeds through release of the substrate and Mg1 followed by lifting of the Gly-rich loop and disengagement of the C-terminal tail. Coupling of these two structural elements with the release of the first metal ion fills in a key step in the catalytic cycle that has been missing and supports an ensemble of correlated conformational states that mediate the full catalytic cycle for a protein kinase.
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Project description:FAD-linked oxidases constitute a class of enzymes which catalyze dehydrogenation as a fundamental biochemical reaction, followed by reoxidation of reduced flavin. Here, we present high-resolution crystal structures showing the flavoenzyme 6-hydroxy-l-nicotine oxidase in action. This enzyme was trapped during catalytic degradation of the native substrate in a sequence of discrete reaction states corresponding to the substrate-reduced enzyme, a complex of the enzyme with the intermediate enamine product and formation of the final aminoketone product. The inactive d-stereoisomer binds in mirror symmetry with respect to the catalytic axis, revealing absolute stereospecificity of hydrogen transfer to the flavin. The structural data suggest deprotonation of the substrate when bound at the active site, an overall binary complex mechanism and oxidation by direct hydride transfer. The amine nitrogen has a critical role in the dehydrogenation step and may activate carbocation formation at the ?-carbon via delocalization from the lone pair to ?* C(?)-H. Enzymatically assisted hydrolysis of the intermediate product occurs at a remote (P site) cavity. Substrate entry and product exit follow different paths. Structural and kinetic data suggest that substrate can also bind to the reduced enzyme, associated with slower reoxidation as compared to the rate of reoxidation of free enzyme. The results are of general relevance for the mechanisms of flavin amine oxidases.
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