2-Chloro-2-phenylethylamine as a mechanistic probe and active site-directed inhibitor of monoamine oxidase from bovine liver mitochondria

The reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B was investigated to study the mechanism of this enzyme and its inactivation by this compound. 2-Chloro-2-phenylethylamine is a substrate with a Km of 30 microM and a turnover number of 80 min-1 at pH 6.5 at 30 degrees C. Incubation of 2-chloro-2-phenylethylamine with the enzyme led to the normal oxidation product, 2-chloro-2-phenylacetaldehyde, but only traces (0.25 mol%) of 2-phenylacetaldehyde, the product anticipated if the oxidation of substrate involved a stabilized carbanion at C-1 and elimination of chloride ion. These data suggest that a carbanion is not a likely intermediate in the oxidation of amines by monoamine oxidase. During the mechanistic studies we noted time-dependent inactivation of monoamine oxidase B by 2-chloro-2-phenylethylamine under both aerobic and anaerobic conditions. Inactivation was not reversible. Aerobically 2-chloro-2-phenylethylamine is oxidized to 2-chloro-2-phenylacetaldehyde which covalently modifies the enzyme (tau 1/2 = 40 min). Benzyl alcohol, a substrate analog, gives substantial protection against inactivation under aerobic conditions (tau 1/2 = 320 min), suggesting that an active site residue is modified. Anaerobic reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B probably proceeds by direct alkylation of an enzyme residue (tau 1/2 = 140 min). Reduction with [3H]NaBH4 of the inactivated enzyme gave from 0 to 0.7 and from 4.5 to 5.6 mol of hydride incorporation for enzyme inactivated anaerobically and aerobically, respectively. The latter results are in agreement with inactivation by unmodified inhibitor and inactivation by oxidized inhibitor for the anaerobic and aerobic reactions, respectively. It is suggested that 2-chloro-2-phenylethylamine or its oxidation product 2-chloro-2-phenylacetaldehyde may serve as an active site affinity reagent for monoamine oxidase.     

Multiplicity of monoamine oxidase: inhibition of mitochondrial monoamine oxidase activity by isopropylhydrazide of D,L-serine]

Isopropylhydrazide of D,L-serine (IHS) inhibits by 50% (at 37 degrees for 10 min) deamination of serotonin or beta-phenylethylamine by monoamine oxidases from bovine brain stem mitochondrial membranes at the 2.6 X X 10(-5) M or 9 X 10(-5) M, respectively. In order to inhibit by 50% the deamination of tyramine under the same conditions a considerably lower (2.5 X X 10(-6) M) concentration of IHS is required. Kinetic studies of inhibition of enzymatic deamination of all the three biogenic monoamines by IHS showed that the irreversible blocking of the monoamine oxidase activity is preceeded by formation of dissociating enzyme-inhibitor complexes. Values of the dissociation constants of these complexes measured (at 37 degrees) with serotonin, phenylethylamine or tyramine as substrates for estimation of the residual monoamine oxidase activity are 0.47; 0.13 or 0.023 mM, respectively. Significant differences are also found between thermodynamic and activation parameters characterizing both both steps of interaction between IHS and the monoamine oxidases of mitochondrial membranes in the experiments with serotonin, phenylethylamine or tyramine as substrates. The data obtained suggest the existence of different monoamine oxidases (or their active sites) catalyzing oxidative deamination of serotonin, phenylethylamine or tyramine in the fragments of mitochondrial membranes from bovine brain stem.     

Neurochemical and neuropharmacological properties of 4-Fluorotranylcypromine

1. The 4-fluoro analogue of the monoamine oxidase-inhibiting antidepressant tranylcypromine was compared to the parent drug with regard to the following: inhibition of monoamine oxidases A and B in vitro and ex vivo; levels of both drugs in brain, liver, and blood after injection of equimolar doses; and effects on brain levels of the amines 2-phenylethylamine, tryptamine, norepinephrine, dopamine, and 5-hydroxytryptamine. 2. 4-Fluorotranylcypromine was found to be 10 times more potent than tranylcypromine at inhibiting monoamine oxidases A and B in vitro in rat brain homogenates. 3. After administration (0.1 mmol/kg, ip), 4-fluorotranylcypromine attained higher brain and liver levels and provided greater availability than did tranylcypromine after the injection of an equimolar amount. 4. At the dose employed, the ex vivo monoamine oxidases A and B inhibitory profiles in brain and liver over a 24-hr period following tranylcypromine and 4-fluorotranylcypromine treatment were not different from each other. 5. Although the drugs had similar effects on inhibition of brain MAO ex vivo, they differed from one another at several time intervals in the increases in concentrations of 2-phenylethylamine, tryptamine, norepinephrine, dopamine, and 5-hydroxytryptamine produced in brain. 6. In conclusion, fluorination of tranylcypromine in the 4 position of the phenyl ring produced a drug which was more potent than the parent drug at inhibiting MAO in vitro and attained higher levels in brain than did tranylcypromine itself after intraperitoneal injection of equimolar amounts of the drugs. 4-Fluorotranylcypromine increased the concentrations of trace amines, catecholamines, and 5-hydroxytryptamine in brain at most time intervals following intraperitoneal injection, and at some time intervals there were differences from tranylcypromine with regard to the amine concentrations produced.     

L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme.

Here we report the cDNA-deduced amino-acid sequence of L-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, D-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl(-), prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.     

Medical implications from the crystal structure of a copper-containing amine oxidase complexed with the antidepressant drug tranylcypromine

The X-ray crystal structure of the copper-containing quinoprotein amine oxidase from E. coli has been determined in complex with the antidepressant drug tranylcypromine to 2.4 A resolution. The drug is a racemic mix of two enantiomers, but only one is seen bound to the enzyme. The other enantiomer is not acting as a substrate for the enzyme as no catalytic activity was detected when the enzyme was initially exposed to the drug. The inhibition of human copper amine oxidases could be a source of side-effects in its use as an antidepressant to inhibit the flavin-containing monoamine oxidases in the brain.     

Specificity of endogenous substrates for types A and B monoamine oxidase in rat striatum

Abstract: Studies were designed to evaluate specificity of the transmitter amines serotonin (5-hydroxytryptamine, 5-HT) and dopamine (DA), as well as the trace amines p -tyramine ( p -TA) and β -phenylethylamine (PEA) for types A and B monoamine oxidase (MAO) in rat striatum. 5-HT was found to be a specific substrate for the type A enzyme. However, the specificity of PEA for the type B enzyme was found to be concentration-dependent. When low concentrations of PEA and 5-HT were used to measure type B and type A activities, respectively, both clorgyline and deprenyl were highly selective for the sensitive form of MAO in vivo. However, as the concentration of PEA was increased, the type B inhibitor deprenyl became less effective in preventing deamination of PEA. Conversely, the type A inhibitor clorgyline became more effective in this regard. Kinetic analysis following selective in vivo inhibition showed PEA deamination by both forms of MAO with a 13-fold greater affinity for the type B enzyme. In vivo dose-response curves obtained with the common substrates DA and p -TA showed approximately 20% deamination by the B enzyme. Kinetic values for DA and p -TA deamination in in vivo -treated tissue possessing only type A or type B MAO activity, revealed a 2.5-fold greater affinity for the type A enzyme. These studies show the importance of concentration on substrate specificity in striatal tissue. The results obtained characterize the common substrate properties of DA and p -TA as well as of PEA in rat striatum. In addition, the presence of regional specificity for 5-HT deamination by only type A MAO is demonstrated.     

On preparative separation of brain mitochondrial monoamine oxidases.

A method was developed for solubilization from bovine brain stem mitochondrial fraction of monoamine oxidases deminating biogenic amines. Preparative separation of the monoamine oxidases, possessing different substrate specificities, was achieved by column chromatography on a biospecific adsorbent AH-Sepharose 4 B. The enzyme preparations thus obtained did not contain any detectable by disc-electrophoresis of isoelectrofocusing in polyacrylamide gels proteins which were devoid of the monoamine oxidase activity.     

On Preparative Separation of Brain Mitochondrial Monoamine Oxidases

A method was developed for solubilization from- bovine brain stem mitochondrial fraction of monoamine oxidases deminating biogenic amines. Preparative separation of the monoamine oxidases, possessing different substrate specificities, was achieved by column chromatography on a biospecific adsorbent AH-Sepharose 4 B. The enzyme preparations thus obtained did not contain any detectable by disc-electrophoresis or isoelectrofocusing in polyacrylamide gels proteins which were devoid of the monoamine oxidase activity.     

Electrical Stimulation of the Substantia Nigra and Changes of 2-Phenylethylamine Synthesis in the Rat Striatum

In rats pretreated with deprenyl (2 mg/kg), electrical stimulation of the left substantia nigra produced an increase in the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the left striatum by 57 and 45%, but the levels of 2-phenylethylamine and p-tyramine decreased by 22 and 41%, respectively, as compared with those in the right striatum. The administration of alpha-methyl-p-tyrosine (1.25 mg/kg, i.p.), a tyrosine hydroxylase inhibitor, 1 h before nigral stimulation, did not affect the concentration of 2-phenylethylamine in unstimulated striata but prevented the stimulation-induced decrease in the concentration of 2-phenylethylamine. Neither stimulation nor alpha-methyl-p-tyrosine affected the activity of monoamine oxidase A or B, and stimulation did not produce any change in striatal blood flow, a finding demonstrating that the changes in the rate of accumulation of 2-phenylethylamine were not due to changes in catabolism or removal of 2-phenylethylamine from the brain. These experiments demonstrate that the rate of synthesis of striatal 2-phenylethylamine is decreased following nigral stimulation and that this effect is blocked after partial inhibition of tyrosine hydroxylase. This suggests that 2-phenylethylamine is present in tyrosine hydroxylase-containing neurons and therefore supports the coexistence of 2-phenylethylamine and dopamine in the nigrostriatal pathway.     

ENZYMATIC ISOTOPIC ASSAY FOR AND PRESENCE OF β-PHENYLETHYLAMINE IN BRAIN

Abstract— An enzymatic isotopic assay for the measurement of β-phenylethylamine in brain, with a sensitivity of 100-200 pg, has been developed. With this assay, the endogenous β-phenylethylamine content (1.5 ng/g) in the rat brain has been determined. Phenylalanine administration increases the brain levels of this amine; inhibition of monoamine oxidase causes a 40-fold increase in brain β-phenylethylamine. After a combined treatment with a monoamine oxidase inhibitor and phenylalanine, the β-phenylethylamine content in the brain increases to about 400-fold. This increase can be blocked by the central decarboxylase inhibitor NSD-1055. p-Chlorophenylalanine also increases β-phenylethylamipe concentration in the brain, and this effect is potentiated by a simultaneous administration of phenylalanine.     

Characterization of amine oxidases from Arthrobacter aurescens and application for determination of biogenic amines

Biogenic amines (BAs) that are produced through naturally occurring decarboxylation of amino acids have toxicological effects on humans. Bacterial amine oxidases are useful tools for the rapid quantification of BAs in foods. To develop amine oxidases for the rapid detection of BAs, the genes for amine oxidases from Arthobacter aurescens TC-1, designated AMAO1, AMAO2, and AMAO3, respectively, were cloned and expressed in Escherichia coli. AMAO1 was catalytically inactive to BAs, and AMAO3 showed a narrow substrate spectrum. In contrast, AMAO2 exhibited activity with relative k _cat/K _M values of 100:49.6:7.6 for 2-phenylethylamine, tyramine, and histamine, respectively. AMAO2 also utilized putrescine and spermidine as substrates, with four or five orders of magnitude lower k _cat/K _M values than that of 2-phenylethylamine. AMAO2 and AMAO3 were seriously affected by substrate inhibition. Using BA mixtures (consisting of 2-phenylethylamine, tyramine, and histamine) as samples, the detection range of the enzymatic analysis of BA using AMAO2 was determined to be 2.5–120 μM, and its detection limit was 2.3 μM. Analysis of five commercial cheese products revealed that the BA contents determined by the enzymatic methods showed a good agreement with the sum of three monoamines and histamine by HPLC. Therefore, the enzymatic assay using AMAO2 can be used in quality control of food products through rapid, sensitive, and preliminary estimation of major BAs including the most important TyrN and HisN in foods.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.     

Structure-based redesign of cofactor binding in putrescine oxidase

Putrescine oxidase (PuO) from Rhodococcus erythropolis is a soluble homodimeric flavoprotein, which oxidizes small aliphatic diamines. In this study, we report the crystal structures and cofactor binding properties of wild-type and mutant enzymes. From a structural viewpoint, PuO closely resembles the sequence-related human monoamine oxidases A and B. This similarity is striking in the flavin-binding site even if PuO does not covalently bind the cofactor as do the monoamine oxidases. A remarkable conserved feature is the cis peptide conformation of the Tyr residue whose conformation is important for substrate recognition in the active site cavity. The structure of PuO in complex with the reaction product reveals that Glu324 is crucial in recognizing the terminal amino group of the diamine substrate and explains the narrow substrate specificity of the enzyme. The structural analysis also provides clues for identification of residues that are responsible for the competitive binding of ADP versus FAD (~50% of wild-type PuO monomers isolated are occupied by ADP instead of FAD). By replacing Pro15, which is part of the dinucleotide-binding domain, enzyme preparations were obtained that are almost 100% in the FAD-bound form. Furthermore, mutants have been designed and prepared that form a covalent 8α-S-cysteinyl-FAD linkage. These data provide new insights into the molecular basis for substrate recognition in amine oxidases and demonstrate that engineering of flavoenzymes to introduce covalent linkage with the cofactor is a possible route to develop more stable protein molecules, better suited for biocatalytic purposes.     

Human acid beta-glucosidase: use of sphingosyl and N-alkyl-glucosylamine inhibitors to investigate the properties of the active site

Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the beta-glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The specificity of binding to the active site of this enzyme was evaluated using series of inhibitors including synthetic sphingosines, N-alkyl(Cn)-deoxynojirimycins (1,5-dideoxy-5-iminoglucose) and N-Cn-glucosylamines. The sphingosines were rapidly reversible inhibitors with maximal potency (IC50 approximately 78-150 micro M) at chain lengths of 14-18 carbons. The presence of unsaturation between C4 and C5 was required for inhibition of enzyme activity. Neither the nature of this bond (double or triple bond) nor the presence of erythro or threo configurations at C2 influenced inhibitory potency. The N-C10- to N-C14-deoxynojirimycins were rapidly reversible inhibitors with Ki approximately 8.5 nM. In comparison, the 1-amino glucose derivatives, i.e., N-Cn-glucosylamines (n = 12-18), were more potent (IC50 approximatley 0.3-3 nM) and their maximal inhibitory potencies were dependent on time as well as enzyme and substrate concentrations: i.e., the N-C12- to N-C18-glucosylamines were competitive, slow-tight binding inhibitors. Analyses of progress curves at various N-Cn-glucosylamine (n = 14-18) concentrations indicated the formation of rapidly dissociating initial EI collison complex which then undergoes a conformational change to a slowly reversible EI complex. These results were consistent with the long chain N-Cn-glucosylamines being reaction intermediate analogues and with this enzyme's hydrolytic mechanism requiring a conformational change during the transition state.     

Human brain monoamine oxidase: solubilization and kinetics of inhibition by octylglucoside

Complete solubilization of both the A and B forms of human brain monoamine oxidase (MAO) occurred when crude mitochondria were incubated in the presence of 50 mM octylglucoside (OG). Upon removal of this nonionic detergent by dialysis, approximately 100% of the starting activity was present in the dialysate. The effects of solubilization were examined by comparison of several properties of the membrane-bound and OG-treated oxidases. The percentage inhibition of phenylethylamine (PEA) and the 5-hydroxytryptamine (5-HT) deamination by deprenyl and clorgyline were identical. The Km values obtained for the deamination of PEA, a B-selective substrate, 5-HT, an A-selective substrate, and tyramine (TYR), a nonselective substrate, were also comparable. OG was found to inhibit type A (I50 = 8.1 mM) and B (I50 = 4.7 mM) MAO activities at concentrations at least 10-fold below those used to solubilize the oxidases. Kinetic studies revealed that OG was an apparent competitive inhibitor of PEA deamination whereas OG produced a mixed-type pattern of inhibition when 5-HT was the variable substrate. Inhibition of TYR deamination by either the A or B form of MAO produced a mixed pattern of inhibition. The findings herein suggest that solubilization of the A and B forms of MAO by OG does not significantly alter the substrate and inhibitor specificity of the oxidases following removal of detergent. However, in the presence of concentrations of OG 50 times less than the critical micellar concentration of this detergent, marked inhibition of deamination by both forms of human brain MAO is observed. Accordingly, the usefulness of OG is limited to situations where the detergent is completely removed before quantitation of MAO activity.     

Characterization of rat pulmonary monoamine oxidase.

The substrate specificities and kinetics of rat lung monoamine oxidase (MAO) have been studied. Utilizing the irreversible MAO inhibitors, clorgyline and deprenyl, rat lung was shown to possess at least two types of MAO, A and B. Tyramine was a substrate for both forms of the enzyme, whereas 5-hydroxytryptamine (5-HT) was a preferred substrate for the A-form. In contrast to most other tissues, 2-phenylethylamine was not solely a B-type substrate for the rat lung MAO and some metabolism by the A-type was apparent $\text{(}\text{B}\text{A}\text{=}\text{80}\text{20}\text{)}$. Using tyramine as substrate the ratio A/B was shown to be $\text{55}\text{45}$. Rat pulmonary MAO-B was inhibited by deprenyl and the kinetics of MAO-A studied. The Km values for the A-form for tyramine, phenylethylamine and 5-HT were relatively similar and were 270, 244 and 170 μM respectively. Whereas, when the A-form was inhibited by clorgyline, the Km values for the B-form were found to differ considerably: 330, 42 and 850 μM for tyramine, phenylethylamine and 5-HT respectively.     

Inhibition of mitochondrial ATPase by water-soluble carbodiimide]

Modification of the carboxyl group with pK 6,8-7,0 of isolated factor F1 by N-cyclohexyl-N'-beta-(4-xethylmorpholine) ethylcarbodiimide (CMCD) leads to the inhibition of the ATPase activity of the enzyme. The reaction rate of factor F1 with CMCD drops in the presence of ATP. It has been shown that during the first stage of the reaction reversible binding of CMCD with factor F1 occurs, forming a stable "enzyme--inhibitor" complex (Kdis=2.10(-4) M). N-cyclohexyl-N'-beta-(4-methylmorpholine) ethylcarbamide, a close analog of CMCD, is a competitive inhibitor of the ATPase reaction with Ki=9.10(-4) M. It is assumed that the carboxyl group, which reacts with CMCD, is located in the catalytic site of factor F1 and serves as the ligand of Me2+ in binding the substrate of the ATPase reaction Me-ATP. The reaction of factor F1, which was modified by CMCD, with proflavine, is accompanied by the covalent binding of the fluorescent label to the enzyme. The binding of proflavine to factor F1 leads to a sharp drop in the solubility of the enzyme in water.     

Oxidation of beta-phenylethylamine by both types of monoamine oxidase: effects of substrate concentration and pH.

β-Phenylethylamine (PEA) was characterized as substrate for both type A and type B monoamine oxidase (MAO) in rat brain mitochondria at different substrate concentrations and at different pHs of the reaction media. The experiments on sensitivity to clorygline and deprenyl showed that the inhibition patterns with PEA as substrate differed markedly at different substrate concentrations: at 10 μM, PEA acted as a specific substrate for type B MAO, but at 50–1000 μM it became a common substrate for both types of MAO. The inhibition patterns were also affected markedly by a small change in pH of the reaction medium, especially when PEA concentrations were 50 and 100 μM: the change in pH from 7.2 to 7.8 resulted in the incresse in the proportion of type A MAO by 20–30 per cent. To investigate the mechanisms of such changes in substrate specificity of PEA, kinetic analyses were carried out at pH 7.2 and 7.8 with the uninhibited, the clorgyline-treated (type B) and the deprenyl-treated (type A) enzyme. The Lineweaver-Burk plots for the uninhibited MAO showed strong substrate inhibition for both pHs, which is more marked at pH 7.8 than at pH 7.2. Pretreatment of the enzyme with 10^?7 M clorgyline resulted in generally similar K_m values for PEA to those of the uninhibited enzyme, and the substrate inhibition at pH 7.8 was also stronger than that at pH 7.2. After pretreatment with 10^?7 M deprenyl, the K_m values were higher and the V_max values were lower than those of the uninhibited or the clorgyline-treated enzyme; there was no or only slight substrate inhibition in these curves. These results suggest that the remarkable changes in substrate specificity observed at different PEA concentrations and at different pHs may be due to the strong substrate inhibition of type B MAO.     

Irreversible Inhibition of Thymidylate Synthase by Pyridoxine (B6) Analogues

Abstract

Three vitamin B₆ analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4′,5′-dichloro-, 4,5′-dibromo- and 4′, 5′-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme: inhibitor affinity complex is formed prior to the irreversible reaction. 4′,5′-Dibromo-pyridoxine exhibited a greater binding affinity (lower K_i) for thymidylate synthase than 4′,5′-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10μM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B₆ analogues are active site-directed inhitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.