Domains in bovine serum amine oxidase

Analysis of the thermal unfolding of bovine serum amine oxidase by differential scanning calorimetry reveals for the dimeric protein a four domain structure consisting of two sets of domains. Each set contains two domains of similar size. The two smaller domains, in contrast with the larger ones, greatly differ in thermostability. Removal of copper changes the calorimetric pattern dramatically. The findings confirm that the metal cofactor plays a structural role. Since the enzyme contains two copper atoms and only one titratable carbonyl group, the calorimetric pattern suggests that the difference in thermostability of the two small domains might be due to the presence of a single organic cofactor.     

Modulation of bovine serum amine oxidase activity by hydrogen peroxide

Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.     

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

1. Cholesteryl 3β-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K_m for cholesteryl sulphate is 500μm and for free cholesterol 50μm under the same conditions. 3. Cholesteryl 3β-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K_i for cholesteryl phosphate 28μm, for cholesteryl sulphate 110μm, for cholesteryl acetate 65μm) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20α-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K_i for pregnenolone is 130μm and K_i for 20α-hydroxycholesterol is 17μm. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K_i16μm). A number of other Δ⁵-3β-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3β-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20α-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3β-Hydroxychol-5-enoic acid, 3α-hydroxy-5β-cholanic acid and 3β-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3β-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.     

Polyamine cytotoxicity in the presence of bovine serum amine oxidase

The toxicity of extracellular spermine, determined in the presence of fetal calf serum, was studied using three cell lines: FM3A, L1210, and NIH3T3 cells. Amine oxidase in fetal calf serum produces aminodialdehyde generating acrolein spontaneously, H(2)O(2), and ammonia from spermine. Spermine toxicity was prevented by aldehyde dehydrogenase, but not by catalase. Similar concentrations of spermine and acrolein were needed to produce toxicity. Other aldehydes (formaldehyde, acetaldehyde, and propionaldehyde) and hydrogen peroxide were less toxic than acrolein. Spermidine and 3-aminopropanal, which produces acrolein, also exhibited severe cytotoxicity. The degree of cytotoxicity of spermine, spermidine, and 3-aminopropanal was nearly parallel with the amount of acrolein produced from each compound. Thus, it was deduced that acrolein is a major toxic compound produced from polyamines (spermine and spermidine) by amine oxidase.     

Crystal structure of amine oxidase from bovine serum

Copper-containing amine oxidase extracted from bovine serum (BSAO) was crystallized and its three-dimensional structure at 2.37A resolution is described. The biological unit of BSAO is a homodimer, formed by two monomers related to each other by a non-crystallographic 2-fold axis. Each monomer is composed of three domains, similar to those of other amine oxidases from lower species. The two monomers are structurally equivalent, despite some minor differences at the two active sites. A large funnel allows access of substrates to the active-site; another cavity, accessible to the solvent, is also present between the two monomers; this second cavity could allow the entrance of molecular oxygen necessary for the oxidative reaction. Some sugar residues, bound to Asn, were still present and visible in the electron density map, in spite of the exhaustive deglycosylation necessary to grow the crystals. The comparison of the BSAO structure with those of other resolved AO structures shows strong dissimilarities in the architecture and charge distribution of the cavities leading to the active-site, possibly explaining the differences in substrate specificity.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone     

Roles of the two copper ions in bovine serum amine oxidase

With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically identical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half-depBSAO were estimated to be 5 X 10(6), 5 X 10(4), and 1 X 10(5) M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of bovine serum amine oxidase with the polyamine oxidase inactivator MDL 72527

MDL 72527 was considered a selective inhibitor of FAD-dependent polyamine oxidases. In the present communication, we demonstrate that MDL 72527 inactivates bovine serum amine oxidase, a copper-containing, TPQ-enzyme, time-dependently at 25 degrees C. In striking contrast, the enzyme remained active after incubation with excessive MDL 72527 at 37 degrees C, even after 70 h of incubation. Inactivation of BSAO with MDL 72527 at 25 degrees C did not involve the cofactor, as was shown by spectroscopy and by reaction with phenylhydrazine. Docking of MDL 72527 is difficult, owing to its size and two lipophilic moieties, and it has been shown that minor changes in reaction rate of substrates cause major changes in K(m) and k(cat)/K(m). We hypothesise that subtle conformational changes between 25 and 37 degrees C impair MDL 72527 from productive binding and prevent the nucleophilic group from reacting with the double bond system.     

Benzylhydrazine as a pseudo-substrate of bovine serum amine oxidase.

Bovine serum amine oxidase is inhibited by benzylhydrazine (BHy), but recovers full activity after a few hours incubation [Hucko-Haas & Reed (1970) Biochem. Biophys. Res. Commun. 38, 396-400]. The first phase of the process, requiring about 15 min, was found to consist of a mechanism-based hydrazine-transfer reaction leading to formation of the hydrazine-bound enzyme, benzaldehyde and H2O2. At variance with the enzymic process, the reaction with O2 preceded the benzaldehyde release. Two reaction intermediates could be characterized by optical spectroscopy and were assigned as the azo derivative and the benzaldehyde hydrazone, the latter one probably being involved in the reaction with O2. No reduction of Cu was detected at any stage. The hydrazine adduct could also be obtained by stoichiometric reaction of hydrazine with the native enzyme. The decay of this species occurred in about 8 h and was not studied in detail. The Cu-binding inhibitor NN-diethyldithiocarbamate affected the BHy reaction by stabilizing the benzaldehyde hydrazone form as against the azo derivative and the reaction with O2. However, under these same conditions the initial spectroscopic properties of the diethyldithiocarbamate adduct were recovered if the oxidase was left overnight. The reaction with O2 was abolished only upon removal of at least one Cu atom from the enzyme. On the basis of the failure to detect any change of Cu redox state and the enzyme behaviour in the presence of inhibitors, a reaction mechanism involving the formation of a hydroperoxy intermediate, as in the FAD-containing enzymes, is tentatively proposed.     

Substrate specificity of polyphenol oxidase

Abstract

The ubiquitous type-3 copper enzyme polyphenol oxidase (PPO) has found itself the subject of profound inhibitor research due to its role in fruit and vegetable browning and mammalian pigmentation. The enzyme itself has also been applied in the fields of bioremediation, biocatalysis and biosensing. However, the nature of PPO substrate specificity has remained elusive despite years of study. Numerous theories have been proposed to account for the difference in tyrosinase and catechol oxidase activity. The “blocker residue” theory suggests that bulky residues near the active site cover CuA, preventing monophenol coordination. The “second shell” theory suggests that residues distant (～8?Å) from the active site, guide and position substrates within the active site based on their properties e.g., hydrophobic, electrostatic. It is also hypothesized that binding specificity is related to oxidation mechanisms of the catalytic cycle, conferred by coordination of a conserved water molecule by other conserved residues. In this review, we highlight recent developments in the structural and mechanistic studies of PPOs and consolidate key concepts in our understanding toward the substrate specificity of PPOs.     

Inactivation of bovine plasma amine oxidase by haloallylamines

Various 2- and 3-haloallylamines were synthesized and evaluated as inhibitors of the quinone-dependent bovine plasma amine oxidase (BPAO). 3-Haloallylamines, which were previously found to be good inhibitors of the flavin-dependent mitochondrial monoamine oxidase (MAO), exhibited a time-dependent inactivation of BPAO, with the 2-phenyl analogs being more potent than the 2-methyl analogs. No plateau of enzyme activity loss was observed, suggestive of a lack of competitive partitioning to normal turnover. The (E)- and (Z)-2-phenyl-3-fluoro analogs were the most potent (low microM IC(50)s), with the corresponding 3-bromo and 3-chloro analogs being >10-fold less potent. In each case, the Z-isomers were more potent than the E-isomers, the reverse of the configurational inhibitory preference observed with MAO. In contrast to the 2-phenyl analogs, 3-phenyl-2(or 3)-chloroallylamines displayed a partitioning behavior, consistent with these being both substrates and inactivators of BPAO.     

Peculiar effects of temperature and polyvinylalcohol on the activity of bovine serum amine oxidase

An inflexion point of enzyme activity at 38 - 42 degrees C of the bovine serum amineoxidase was found. This result, associated with non-strict Arrhenius curves and slightly different activation energies in various temperature intervals, suggests some conformational transitions at the mentioned temperatures. The high molecular weight polyvinylalcohol (100,000 Da) generated an activatory effect and a sigmoidal (non-Michaelis) curve of the dependence of the activity on the substrate concentrations, while the low molecular weight polyvinylalcohol (20,000 Da) does not produce this effect. The different ratio of the two types of polyvinylalcohol/enzyme monomer sizes is considered to be responsible for these different effects on the enzyme kinetics.     

Substrate specificity of copper-containing plant amine oxidases

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.     

Substrate specificity of ascorbate oxidase.

Ascorbate oxidase oxidizes leuco 2, 6-dichloroindophenol to the blue quinoid dye and produces spectral changes in the UV spectra of certain substituted polyhydric and amino phenols at pH 5.7. The new peaks produced by the addition of enzyme to the dichlorohydroquinones (2,5 and 2,6) and hydroxyhydroquinone correspond to the respective p-quinones of these substrates. At pH 5.7, the enzyme does not oxidize hydroquinone, barely oxidizes chlorohydroquinone, but oxidizes 2,6- and 2,5-dichlorohydroquinone and hydroxyhydroquinone at a rate about $\text{1}\text{12}$ that of ascorbic acid, with the uptake of one gram atom of oxygen per mole of substrate. A correlation has been found between the concentration of anion present in solution at pH 5.7 and the rate of oxidation of compounds of the hydroquinone series by the enzyme. The results indicate that an anionic form of the substrate is an important requirement of the enzyme specificity.     

Substrate specificity of peroxisomal acyl-coA oxidase

Using peroxisomes as an enzyme source, we have determined the K_m and V_max values of two plant peroxisomal acyl-CoA oxidases for various acyl-CoAs. Plant peroxisomal acyl-CoA oxidases seem to be active on long and short acyl-chain CoAs.     

Purification of bovine plasma amine oxidase

A new method for the purification of bovine plasma amine oxidase is described. The enzyme is purified by ammonium sulfate precipitation and by affinity chromatography performed with AH-Sepharose 4B and concanavalin A-Sepharose. Three activity peaks were separated, all showing similar properties. Specific activity is the highest described for this enzyme. The enzyme appears to contain 2 copper atoms and 1 carbonyl group/molecule.