An investigation of bovine serum amine oxidase active site stoichiometry: evidence for an aminotransferase mechanism involving two carbonyl cofactors per enzyme dimer

Janes, Susan; Klinman, Judith P.

doi:10.1021/bi00232a034

Cited by 124 publications

(109 citation statements)

References 32 publications

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“…45). Formation of the phenylhydrazone during phenylhydrazine titration of AO has been monitored by an increase in absorbance at 450 nm (46). However, monitoring of phenylhydrazine binding to PSII by this approach is not possible due to spectral interference from pigments associated with PSII (47,48).…”

Section: Discussionmentioning

confidence: 99%

Probing the Structure of Photosystem II with Amines and Phenylhydrazine

Anderson

Ouellette

Barry

2000

Journal of Biological Chemistry

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-[14 C]butylamine and phenylhydrazine were employed as probes. Neither compound can be oxidized by a transamination or addition/elimination mechanism, but both can react with activated carbonyl groups, produced as a result of posttranslational modification of amino acid residues, to give amine-derived adducts.14 C incorporation into the PSII subunits D2/D1 and CP47 was obtained upon treatment of PSII with either t-[ 14 C]butylamine or [ 14 C]phenylhydrazine. For t-butylamine and methylamine, the amount of labeling increased when PSII was treated with denaturing agents. Labeling of CP47, D2, and D1 with methylamine and phenylhydrazine approached a one-to-one stoichiometry, assuming that D2 and D1 each have one binding site. Evidence was obtained suggesting that reductive stabilization and/or access are modulated by PSII light reactions. These results support the proposal that PSII subunits D2, D1, and CP47 contain quinocofactors and that access to these sites is sterically limited.Photosystem II (PSII), 1 a membrane-bound protein complex, catalyzes the light-driven oxidation of water and the reduction of plastoquinone. PSII consists of hydrophobic and extrinsic subunits and contains chlorophyll, plastoquinone, manganese, and several other bound cofactors. Water oxidation occurs at a manganese-containing catalytic site located on the lumenal side of the membrane. After photoexcitation, the primary chlorophyll donor, P 680 , transfers an electron from its excited state to a pheophytin molecule. Pheophytin transfers the electron to a quinone acceptor molecule, Q A , which in turn reduces a second quinone acceptor, Q B (reviewed in Refs. 1 and 2). PSII contains two well characterized redox-active tyrosines, Z and D (3, 4). Z, an intermediate electron carrier between P 680 and the manganese cluster, reduces oxidized P 680 (5). D forms a stable radical and has no known role in water oxidation (6). Oxidation of a third, posttranslationally modified tyrosine, M, occurs in PSII site-directed mutants (6 -9).Primary amines are known to be substrate analogs and inhibitors of oxygen evolution (10 -12). Recently, the covalent incorporation of 14 C-labeled primary amines into PSII subunits has been observed under reducing conditions (13). Evidence for oxidation of [ 14 C]benzylamine by PSII was obtained by detection and quantitation of [ 14 C]benzaldehyde (13). Because PSII exhibits this amine oxidation activity and an aldehyde product has been observed, covalent incorporation of 14 C from labeled primary amines into PSII subunits could be caused by two possible reactions (13). In the first possible reaction, addition of reductant traps a Schiff base complex of substrate ( 14 C-labeled amine) and an amino acid side chain that has been posttranslationally modified to contain an activated carbonyl group (13,14,15). In the second possible reaction, addition of reductant traps a Schiff base complex of product ( 14 C-labeled aldehyde) and the amino group of a lysine side chain (13). When formaldehyde is produced, this reaction produ...

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Section: Discussionmentioning

confidence: 99%

Probing the Structure of Photosystem II with Amines and Phenylhydrazine

Anderson

Ouellette

Barry

2000

Journal of Biological Chemistry

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“…V/cZ4, where, V is reaction volume (1.0 ml) and E , ,~ = 3.2 mM-' . cm-' is the molar absorption coefficient for diethylpyrocarbonate-modified histidyl residue [17]). (B) AO-J ( 0 ) (11 pM) and AO-JJ (+) (11 pM) show maximum absorbance of 0.450 at 310 nm ( E~~~ = 40.9 mM-' .…”

Section: Determination Of 14-diamino-2-butyne-binding Residuementioning

confidence: 99%

Active‐Site Covalent Modifications of Quinoprotein Amine Oxidases from Aspergillus niger

Frébort¹,

Peč

Luhová

et al. 1994

European Journal of Biochemistry

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Interactions of two distinct quinoprotein amine oxidases from Aspergillus niger, AO-I and AO-11, with active-site covalent modifiers have been investigated. Both enzymes are inhibited similarly by phenylhydrazine or p-nitrophenylhydrazine, forming an orange Schiff base with a carbonyl group of topaquinone cofactor. Modification of histidyl and tyrosyl residues by diethylpyrocarbonate and sulfhydryl groups by 5,5'-dithio-bis-(2-nitrobenzoic acid) and 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole have been described. A substrate analog, 1,4-diamino-2-butyne, was found to function as a mechanism-based inhibitor. It shows both substrate saturation kinetics and time-dependent irreversible inhibition caused by formation of pynole bound to the active site. The pyrrole formation was confirmed spectrophotometrically by reaction with Ehrlich's reagent at 525 nm. Inhibition by 1,4-diamino-2-butyne produces a new maximum in the absorption spectra of AO-I and AO-I1 at 310 nm and 306 nm, respectively. Inactivated AO-I was digested by proteases ; labeled peptides were purified by C18 HPLC and sequenced by Edman degradation. Data reveal the evidence that 1P-diamino-2-butyne reacts with the &-amino group of the Lys356 residue in the sequence Lys-Met-Pro-AsnAla of Aspergillus niger amine oxidase AO-I.Amine oxidase of Aspergillus niger is a copper-carbonyl enzyme, which was found in the mid 1960s, purified and crystallized [l, 21. Copper and an undetermined carbonyl cofactor were found to function in the catalytic cycle [2-41. Nowadays, the importance of the fungal amine oxidase has risen again because the enzyme can be applied to monitor histamine contents in fishery products [5]. A massive production of the enzyme on the industrial scale has increased current interest.Recently, we have found two amine oxidases (EC 1.4.3.6), named AO-I and AO-11, in Aspergillus niger mycelia grown on n-butylamine medium [6, 71. AO-I is the enzyme previously reported, consisting of two 75-kDa subunits, while AO-I1 is a novel enzyme of 80 kDa. These amine oxidases do not show big differences in their peptide maps, but differ in their N-terminals. They also do not show any remarkable difference in substrate specificity or sensitivity to inhibitors. The cofactors of both enzymes give a typical reaction with p-nitrophenylhydrazine [6, 71 like other topaquiCorrespondence to I. Frkbort, Laboratory of Applied Microbiology, Faculty of Agriculture, Yarnaguchi University, 1 677-1 Yoshida, Yamaguchi, Japan 753Abbreviations. AO-I and AO-11, Aspergillus niger amine oxidases ; Nbs,, 5,5'-dithio-bis-(2-nitrobenzoic acid) ; buffer A, 0.01 M potassium phosphate pH 7.0.Enzymes. Amine oxidase, arnine:O, oxidoreductase (dearninating) (copper containing) (EC 1.4.3.6) ; peroxidase from horseradish (EC 1.11.1.7); pronase P from Streptomyces griseus (EC 3.4.24.31); thermolysin from Bacillus themolyticus rokko (EC 3.4.24.27 It is very probable that AO-I was purified and crystallized in earlier studies but fractions containing AO-I1 were not purified further because of its lower...

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“…Benzylamine is the best substrate for benzylamine oxidase from pig or beef plasma (Janes and Klinman, 1991;Morpurgo et al, 1991). Also, ELAO was able to oxidize benzylamine, with a K m value of 0.4 mm, which is similar to that found for putrescine (Rinaldi et al, 1982).…”

Section: Reaction Of Elao With Benzylaminementioning

confidence: 60%

“…It is reasonable to assume that both reduced enzyme species may react with oxygen to release hydrogen peroxide and ammonia and to regenerate the Cu(II)-quinone species, but at least in the plant enzymes, the radical seems to be much more reactive than the Cu(II)-aminoresorcinol . Although discrepancies concerning the active TPQ:protein stoichiometry are present in the literature (Klinman and Mu, 1994;Agostinelli et al, 1997), most recent studies support an active TPQ:monomer ratio of 1:1 (Janes and Klinman, 1991;Padiglia et al, 1992;McGuirl et al, 1994). …”

Section: CMmentioning

confidence: 99%

Characterization of Euphorbia characias Latex Amine Oxidase1

et al. 1998

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A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo-and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 ؋ 10 M ؊1 cm؊1 and 6000 M ؊1 cm ؊1 , respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinonecontaining amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.Copper-amine oxidases (amine oxygen oxidoreductase deaminating, copper containing; EC 1.4.3.6) are widespread enzymes oxidizing primary amines with the formation of the corresponding aldehyde, ammonia, and hydrogen peroxide: R-CH 2 -NH 2 ϩO 2 ϩH 2 O3 R-CHOϩNH 3 ϩH 2 O 2 .These enzymes are ubiquitous in nature, occurring in microorganisms (fungi and bacteria; Cooper et al., 1992), plants (Medda et al., 1995a), and mammals (McIntire and Hartmann, 1993). The crystal structures of copper-amine oxidases from Escherichia coli (Parson et al., 1995) and pea seedlings (Kumar et al., 1996) were recently determined. Amine oxidases are homodimers of 70-to 95-kD subunits. Each subunit contains a tightly bound Cu(II) center that is essential for the enzyme redox activity (Dooley et al., 1991;Medda et al., 1995b), and TPQ is formed by a posttranslational modification from a tyrosinyl residue in a copperdependent, autocatalytic reaction (Janes et al., 1990;

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An investigation of bovine serum amine oxidase active site stoichiometry: evidence for an aminotransferase mechanism involving two carbonyl cofactors per enzyme dimer

Cited by 124 publications

References 32 publications

Probing the Structure of Photosystem II with Amines and Phenylhydrazine

Probing the Structure of Photosystem II with Amines and Phenylhydrazine

Active‐Site Covalent Modifications of Quinoprotein Amine Oxidases from Aspergillus niger

Characterization of Euphorbia characias Latex Amine Oxidase1

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