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Studies on the active site of pig plasma amine oxidase.

ABSTRACT: 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.

SUBMITTER: Collison D

PROVIDER: S-EPMC1133638 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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Unique protonation states of aspartate and topaquinone in the active site of copper amine oxidase.

Project description:The oxidative deamination of biogenic amines, crucial in the metabolism of a wealth of living organisms, is catalyzed by copper amine oxidases (CAOs). In this work, on the ground of accurate molecular modeling, we provide a clear insight into the unique protonation states of the key catalytic aspartate residue Asp298 and the prosthetic group of topaquinone (TPQ) in the CAO of Arthrobacter globiformis (AGAO). This provides both extensions and complementary information to the crystal structure determined by our recent neutron diffraction (ND) experiment. The hybrid quantum mechanics/molecular mechanics (QM/MM) simulations suggest that the ND structure closely resembles a state in which Asp298 is protonated and the TPQ takes an enolate form. The TPQ keto form can coexist in the fully protonated state. The energetic and structural analyses indicate that the active site structure of the AGAO crystal is not a single state but rather a mixture of the different protonation and conformational states identified in this work.

| S-EPMC9057271 | biostudies-literature

Electron nuclear double-resonance (ENDOR) spectroscopy of amine oxidase from pig plasma.

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.

| S-EPMC1147029 | biostudies-other

Active-sitve titration of pig plasma benzylamine oxidase with phenylhydrazine.

Project description:Pig plasma benzylamine oxidase is a protein containing cupric copper and pyridoxal phosphate. The pyridoxal phosphate is stably linked to the enzyme. Discrepancies in the numbers of active sites per molecule of enzyme are reported in the literature. This paper shows that the fully active pure enzyme contains 3 mol of pyridoxal phosphate per mol, whereas enzymes with a lower specific activity are shown by titration with phenylhydrazine to have a lower pyrdoxal phosphate content.

| S-EPMC1165226 | biostudies-other

Kinetic and spectroscopic studies of bicupin oxalate oxidase and putative active site mutants.

Project description:Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.

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The kinetics of ammonia release during the catalytic cycle of pig plasma amine oxidase.

Project description:The time course of NH3 release during the catalytic cycle of pig plasma amine oxidase was followed by using the quenched-flow technique in conjunction with a sensitive assay for NH3. These studies were made under both air and O2-saturating conditions. The results establish unequivocally that NH3 is released in the step whereby a reduced enzyme species is re-oxidized by molecular O2 rather than in the step leading to the reduced enzyme. It is concluded that the catalytic cycle of the enzyme conforms to an aminotransferase mechanism rather than one in which an imine is an intermediate.

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Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase.

Project description:The flavoprotein d-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant d-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) Angew. Chem., Int. Ed. 53, 4428-4431]. We describe here the use of pH and kinetic isotope effects with (R)-?-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the same catalytic mechanism as the wild-type enzyme. The effects of pH on the steady-state and rapid-reaction kinetics establish that the neutral amine is the substrate, while an active-site residue, likely Tyr224, must be uncharged for productive binding. There is no solvent isotope effect on the kcat/Km value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the kcat/Km value is pH-independent, with an average value of 5.3, similar to values found with amino acids as substrates for the wild-type enzyme and establishing that there is no commitment to catalysis with this substrate. The kcat/KO2 value is similar to that seen with amino acids as the substrate, consistent with the oxidative half-reaction being unperturbed by the mutation and with flavin oxidation preceding product release. All of the data are consistent with the mutant enzyme utilizing the same mechanism as the wild-type enzyme, transfer of hydride from the neutral amine to the flavin.

| S-EPMC5472355 | biostudies-literature

Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity.

Project description:The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

| S-EPMC2927706 | biostudies-literature

Characterization of active site residues of nitroalkane oxidase.

Project description:The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.

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Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase.

Project description:We report a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA), which displays extremely tight binding to BPAO (K(m) 183 +/- 14 nM) and yet is metabolized fairly quickly (k(cat) 0.690 +/- 0.010 s(-1)), with the aldehyde turnover product (2-(6-formylnaphthalen-2-yloxy)ethyl)trimethylammonium serving as a real time reporting fluorophore of the enzyme activity. This allowed for the development of a fluorometric noncoupled assay that is 2 orders of magnitude more sensitive than the spectrophotometric benzylamine assay. The discovery of ANETA involved elaboration of the lead compound 6-methoxy-2-naphthalenemethaneamine by structure-based design, which recognized the ancillary cation binding site of BPAO as the most significant structural features controlling binding affinity. Structure-based design further ensured a high level of selectivity: ANETA is a good substrate of BPAO but is not a substrate of either porcine kidney diamine oxidase (pkDAO) or rat liver monoamine oxidase (MAO-B). ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to respond directly to the enzyme activity in real time.

| S-EPMC2659760 | biostudies-literature

Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper Site in Topaquinone Biogenesis.

Project description:Copper-dependent amine oxidases produce their redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), via the CuII-catalyzed oxygenation of an active site tyrosine. This study addresses possible mechanisms for this biogenesis process by presenting the geometric and electronic structure characterization of the CuII-bound, prebiogenesis (preprocessed) active site of the enzyme Arthrobacter globiformis amine oxidase (AGAO). CuII-loading into the preprocessed AGAO active site is slow ( kobs = 0.13 h-1), and is preceded by CuII binding in a separate kinetically favored site that is distinct from the active site. Preprocessed active site CuII is in a thermal equilibrium between two species, an entropically favored form with tyrosine protonated and unbound from the CuII site, and an enthalpically favored form with tyrosine bound deprotonated to the CuII active site. It is shown that the CuII-tyrosinate bound form is directly active in biogenesis. The electronic structure determined for the reactive form of the preprocessed CuII active site is inconsistent with a biogenesis pathway that proceeds through a CuI-tyrosyl radical intermediate, but consistent with a pathway that overcomes the spin forbidden reaction of 3O2 with the bound singlet substrate via a three-electron concerted charge-transfer mechanism.

| S-EPMC6568004 | biostudies-literature

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