Stereochemistry of 2-phenylethylamine oxidation catalyzed by bacterial copper amine oxidase

The stereochemical course of the reaction catalyzed by a copper amine oxidase from Arthrobacter globiformis has been investigated using 2-phenylethylamine stereospecifically deuterium-labeled at the C1 position. Measurements of deuterium content in the product, phenylacetaldehyde, by gas chromatography-mass spectrometry revealed stereospecific abstraction of the pro-S hydrogen during the enzymatic oxidation, as predicted from the structure modeling for the enzyme-bound substrate.     

pH and deuterium kinetic isotope effects studies on the oxidation of choline to betaine-aldehyde catalyzed by choline oxidase

The FAD-dependent choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate. The enzyme is capable of accepting either choline or betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to betaine-aldehyde and of betaine-aldehyde to glycine-betaine. In the present study, pH and deuterium kinetic isotope effects with [1,2-2H(4)]-choline were used to study the mechanism of oxidation of choline to betaine-aldehyde. The V/K and V(max) pH-profiles increased to limiting values with increasing pH, suggesting the presence of a catalytic base essential for catalysis at the enzyme active site. From the V/K pH-profile with [1,2-2H(4)]-choline, a pK(a) of 8.0 was determined for the catalytic base. This pK(a) was shifted to 7.5 in the V/K pH-profile with choline, indicating a significant commitment to catalysis with this substrate. In agreement with this conclusion, the D(V/K) values decreased from a limiting value of 12.4 below pH 6.5 to a limiting value of 4.1 above pH 9.5. The large D(V/K) values at low pH are consistent with carbon-hydrogen bond cleavage of choline being nearly irreversible and fully rate-limiting at low pH. Based on comparison of amino acid sequences and previous structural and mechanistic studies on other members of the GMC oxidoreductase superfamily, the identity of the catalytic base of choline oxidase is proposed.     

pH dependence of deuterium isotope effects and tritium exchange in the bovine plasma amine oxidase reaction: a role for single-base catalysis in amine oxidation and imine exchange

The pH dependence of steady-state parameters for [1,1-1H2]- and [1,1-2H2]benzylamine oxidation and of tritium exchange from [2-3H]dopamine has been measured in the bovine plasma amine oxidase reaction. Deuterium isotope effects on kcat/Km for benzylamine are observed to be constant, near the intrinsic value of 13.5, over the experimental pH range, indicating that C-H bond cleavage is fully rate limiting for this parameter. As a consequence, pKa values derived from kcat/Km profiles, 8.0 +/- 0.1 (pK1) and 9.0 +/- 0.16 (pKs), can be ascribed to microscopic pKa values for the ionization of an essential active site residue (EB1) and substrate, respectively. Profiles for kcat and Dkcat show that EB1 undergoes a perturbation from 8.0 to 5.6 +/- 0.3 (pK1') in the presence of substrate; additionally, a second ionization, pK2 = 7.25 +/- 0.25, is observed to mediate but not be essential for enzyme reoxidation. The pH dependence of the ratio of tritium exchange to product formation for dopamine also indicates base catalysis with a pKexch = 5.5 +/- 0.01, which is within experimental error of pK1'. We conclude that the data presented herein support a single residue catalyzing both substrate oxidation and exchange, consistent with recent stereochemical results that implicate a syn relationship between these processes [Farnum, M., & Klinman, J.P. (1985) Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1055]. This conclusion contrasts with earlier kinetic data in support of a large rate differential for the exchange of hydrogen from C-1 vs. C-2 of phenethylamine derivatives [Palcic, M.M., & Klinman, J.P. (1983) Biochemistry 22, 5957-5966].(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

pH and kinetic isotope effects on sarcosine oxidation by N-methyltryptophan oxidase

N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of secondary amino acids such as N-methyltryptophan or N-methylglycine (sarcosine). MTOX is one of several flavin-dependent amine oxidases whose chemical mechanism is still debated. The kinetic properties of MTOX with the slow substrate sarcosine were determined. Initial rate data are well-described by the equation for a ping-pong kinetic mechanism, in that the V/K(O)()2 value is independent of the sarcosine concentration at all accessible concentrations of oxygen. The k(cat)/K(sarc) pH profile is bell-shaped, with pK(a) values of 8.8 and about 10; the latter value matches the pK(a) value of the substrate nitrogen. The k(cat) pH profile exhibits a single pK(a) value of 9.1 for a group that must be unprotonated for catalysis. There is no significant solvent isotope effect on the k(cat)/K(sarc) value. With N-methyl-(2)H(3)-glycine as the substrate, there is a pH-independent kinetic isotope effect on k(cat), k(cat)/K(sarc), and the rate constant for flavin reduction, with an average value of 7.2. Stopped-flow spectroscopy with both the protiated and deuterated substrate failed to detect any intermediates between the enzyme-substrate complex and the fully reduced enzyme. These results are used to evaluate proposed chemical mechanisms.     

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.     

Stereochemistry of copper amine oxidase reactions

The stereochemical course of the oxidation of stereospecifically deuterated dopamine and tyramine, catalyzed by porcine plasma amine oxidase, has been investigated using 1H NMR spectroscopy. The oxidation proceeds with loss of the pro-R hydrogen at C-1. This stereochemistry is in contrast to that observed with the analogous copper containing oxidases isolated from pea seedlings (pro-S) and bovine plasma (nonstereospecific). There is no precedent for these three distinct stereochemical reaction courses to be followed by enzymes in the same class. Mechanistic differences among the three enzymes are evident from the profiles of solvent exchange into reaction products; however, these differences cannot account for the overall differential stereochemical courses observed.     

pH and kinetic isotope effects in d-amino acid oxidase catalysis.

The effects of pH, solvent isotope, and primary isotope replacement on substrate dehydrogenation by Rhodotorula gracilis d-amino acid oxidase were investigated. The rate constant for enzyme-FAD reduction by d-alanine increases approximately fourfold with pH, reflecting apparent pKa values of approximately 6 and approximately 8, and reaches plateaus at high and low pH. Such profiles are observed in all presteady-state and steady-state kinetic experiments, using both d-alanine and d-asparagine as substrates, and are inconsistent with the operation of a base essential to catalysis. A solvent deuterium isotope effect of 3.1 +/- 1.1 is observed on the reaction with d-alanine at pH 6; it decreases to 1.2 +/- 0.2 at pH 10. The primary substrate isotope effect on the reduction rate with [2-D]d-alanine is 9.1 +/- 1.5 at low and 2.3 +/- 0.3 at high pH. At pH 6.0, the solvent isotope effect is 2.9 +/- 0.8 with [2-D]d-alanine, and the primary isotope effect is 8.4 +/- 2.4 in D2O. Thus, primary and solvent kinetic isotope effects (KIEs) are independent of the presence of the other isotope, i.e. the 'double' kinetic isotope effect is the product of the individual KIEs, consistent with a transition state in which rupture of the two bonds of the substrate to hydrogen is concerted. These results support a hydride transfer mechanism for the dehydrogenation reaction in d-amino acid oxidase and argue against the occurrence of any intermediates in the process. A pKa,app of approximately 8 is interpreted to arise from the microscopic ionization of the substrate amino acid alpha-amino group, but also includes contributions from kinetic parameters.     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

Reductive trapping of substrate to bovine plasma amine oxidase

Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O2 to H2O2. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation between a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of [14C]benzylamine and [3H]NaCNBH3. The use of the relatively weak reductant, NaCNBH3, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH3 in the presence of substrate, leading to the incorporation of 1 mol of [14C]benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with [3H]NaCNBH3, analogous to an earlier study with [3H]NaCNBH4 (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.     

Inhibition of bovine plasma semicarbazide-sensitive amine oxidase by caffeine

Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes the oxidative deamination of endogenous and exogenous primary amines. SSAO exists in mammals both as a plasma-soluble and as a membrane-bound form, and its active site is able to come into contact with numerous xenobiotic, amine-containing compounds. The kinetic studies performed in this work showed that caffeine inhibition of bovine serum amine oxidase was noncompetitive when benzylamine was used as substrate and mixed when the substrate used was methylamine. Since caffeine contains an imidazole ring, it cannot be excluded that it might bind to an inhibitory imidazoline-binding site on SSAO.     

Effect of phosphate ion on the activity of bovine plasma amine oxidase.

The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 +/- 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 +/- 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.     

Isolation of amine oxidase from bovine plasma by a two-step procedure.

A novel method for isolation of amine oxidase from bovine plasma is reported; it involves a two-step procedure, namely ammonium sulfate fractionation and affinity chromatography with elution by aniline, which is a competitive inhibitor of the enzyme. A homogeneous enzyme, characterized by a specific activity of 0.44 U/mg, was obtained with a yield higher than 50%.     

Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects

Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.     

Insights into the mechanism of flavoprotein-catalyzed amine oxidation from nitrogen isotope effects on the reaction of N-methyltryptophan oxidase

The mechanism of N-methyltryptophan oxidase, a flavin-dependent amine oxidase from Escherichia coli, was studied using a combination of kinetic isotope effects and theoretical calculations. The 15(kcat/Km) kinetic isotope effect for sarcosine oxidation is pH-dependent with a limiting value of 0.994-0.995 at high pH. Density functional theory calculations on model systems were used to interpret these isotope effects. The isotope effects are inconsistent with proposed mechanisms involving covalent amine-flavin adducts but cannot by themselves conclusively distinguish between some discrete electron-transfer mechanisms and a direct hydride-transfer mechanism, although the latter mechanism is more consistent with the energetics of the reaction.     

Highly potent propargylamine and allylamine inhibitors of bovine plasma amine oxidase

Propargylamine was reported many years ago to be a mechanism-based inhibitor of bovine plasma amine oxidase (BPAO), though the potency was modest and allylamine was a substrate. Herein, selected 3-substituted propargylamines and allylamines were found to be potent time-dependent inactivators of BPAO, exhibiting IC(50) values of 2-13 microM at 30 degrees C, making them the most potent BPAO inhibitors reported to date. The most potent compound, trans-3-chloroallylamine, was previously found not to inhibit the flavin-dependent monoamine oxidase (the cis isomer did), and thus appears to be a highly selective inhibitor.     

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (k(red)/K(s), reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pK(a) of the free amine is above 9.3 for all substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the enzyme active site. This perturbation of the substrate amine pK(a) on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme-substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.