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R B Silverman

Publications and source records attributed to R B Silverman.

At least 55 records · Page 3Linked to original sources
PubMed1996

Slow-binding inhibition of gamma-aminobutyric acid aminotransferase by hydrazine analogues.

(3-Hydroxybenzyl)hydrazine and methylhydrazine have been found to be potent slow-binding inhibitors of the pyridoxal 5-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid aminotransferase (GABA-AT). Both compounds follow mechanism A (Morrison, J.F.; Walsh, C. T. Adv. Enzymol. 1988, 61, 201-301) which does not involve formation of a rapidly reversible enzyme-inhibitor complex before the formation of the final tight complex. The rate constant for formation of the enzyme-inhibitor complex determined from the slow-binding kinetics was 2.08 x 10(3) and 1.98 x 10(4) M-1 min-1 for methylhydrazine and (3-hydroxybenzyl)hydrazine, respectively. The rate constant for dissociation of the enzyme--inhibitor complex determined for the slow-binding kinetics was 4.6 x 10(-3) and 5 x 10(-3) min-1, respectively. The inhibition constants calculated from the slow-binding inhibition kinetics are 2.2 microM for methylhydrazine and 0.3 microM for (3-hydroxybenzyl)hydrazine. Reactivation of the inhibited enzyme was not first order, perhaps due to a side reaction of the hydrazine, but was consistent with the results obtained from the slow-binding kinetics. Inhibition constants were calculated from the level of enzyme activity at equilibrium inhibition. These constants are 2.8 and 0.46 microM for methylhydrazine and (3-hydroxybenzyl)hydrazine, respectively, in good agreement with those calculated from the slow-binding inhibition kinetics. 3-Hydrazinopropionate also behaved as a slow-binding inhibitor. However, the dependence of its kinetics on the concentration of inhibitor could not be described by the slow-binding or slow, tight-binding inhibition models. These kinetics could not be described by the tight-binding character of the inhibition because the addition of the competitive inhibitor propionic acid at 100 times its Ki did not affect the shape of the curve for inhibitor concentration dependence. The slow-binding inhibition appeared to require 2-4 molecules of 3-hydrazinopropionate/enzyme. The reactivation of enzyme inhibited by 3-hydrazinopropionate was first order with a rate constant of 6.9 x 10(-3) min-1. Its equilibrium inhibition constant was calculated to be < 20 nM. However, the inhibition constant calculated was dependent on the concentration of inhibitor because of the unusual character discussed above and may be much lower. Only 1 PLP/enzyme dimer reacted with methylhydrazine or (3-hydroxybenzyl)hydrazine, as indicated by Scatchard plots, or with 3-hydrazinopropionate, as shown by a spectrophotometric titration. Slow-binding inhibition does not appear to be the result of a significant enzyme conformational change because there is no change in the tryptophan fluorescence of GABA-AT upon binding either methylhydrazine or 3-hydrazinopropionate. Implications for the design of hydrazine inhibitors of GABA-AT are discussed.

4-Aminobutyrate Transaminase↗
PubMed1996

Direct continuous fluorometric assay for monoamine oxidase B.

The first direct and continuous fluorometric assay for monoamine oxidase B (MAO B) has been developed. E-2,5-Dimethoxycinnamylamine hydrochloride was designed and synthesized and was found to be an excellent substrate for MAO B (Km = 218 microM, Kcat = 435 min-1). This compound has an intense purple fluorescence when irradiated at lambda ex = 343 nm (lambda em = 393 nm) in Tris buffer, pH 9.0, or sodium phosphate buffer, pH 7.2, but under the same conditions, the corresponding aldehyde, the product of the MAO-catalyzed oxidation of E-,5-dimethoxycinnamylamine hydrochloride, does not fluoresce. The activity of MAO B, therefore, can be determined efficiently and rapidly by continuously following the decrease in fluorescence at 393 nm at enzyme concentrations as low as 100 nM. The change in fluorescence is linear up to a substrate concentration of 500 microM.

Animals↗
PubMed1996

Mechanism-based inactivation of gamma-aminobutyric acid aminotransferase by 3-amino-4-fluorobutanoic acid.

The mechanism of inactivation of the pyridoxal 5'-phosphate (PLP)-dependent enzyme gamma-aminobutyric acid (GABA) aminotransferase by 3-amino-4-fluorobutanoic acid (2) has been investigated. As in the case of the homologue, 4-amino-5-fluoropentanoic acid (1), 2 equiv of radiolabeled inactivator become covalently attached to the enzyme, and no transamination, as determined by the lack of conversion of [1-14C] alpha-ketoglutarate into [1-14C] glutamate during inactivation, was observed. In the case of 1, the conclusion was that inactivation was completely the result of modification of the coenzyme and that there was no metabolic turnover; every enzyme molecule catalysed the conversion of one molecule of inactivator to the activated species, which inactivated the enzyme by an enamine mechanism. With 2, however, 6.7 +/- 0.7 equiv of fluoride ions were released during inactivation, and it took 7.6 +/- 0.7 inactivator molecules to inactivate each enzyme dimer. Since no transamination was occurring, another metabolic event besides inactivation must result from the PLP form of the enzyme. Inactivation of GABA amino-transferase with [1,2-14C]-2 produced [14C] acetoacetic acid (about 5.5 equiv) as the metabolite. The 1.93 +/- 0.25 equiv of radioactivity covalently bound to the enzyme after inactivation with [1,2-14C]-2 and gel filtration were completely released by base treatment. HPLC analysis showed that three radioactive compounds, identified as 2, the product of reaction of PLP with acetone (3), and the product of reaction of PLP with acetoacetate (4), were detected. The release of 3 and 4 and the prevention of release of radioactivity by treatment with sodium borohydride are consistent with the formation of covalent intermediates that have beta-carbonyl-like character, such as 6 and/or 7 (Scheme 2). Inactivation of [3H] PLP-reconstituted GABA aminotransferase with 2 followed by gel filtration then base denaturation released all of the radioactivity as a mixture of PLP, 3, and 4. Inactivation with [1,2-14C]-2 resulted in the release of 1.37 equiv of 14CO2, which was shown to be the result of decarboxylation of the acetoacetate/4 after release from the enzyme. These results are not consistent with a Michael addition mechanism (Scheme 3), but are consistent with inactivation by an enamine mechanism; release of the enamine five out of seven turnovers accounts for the formation of acetoacetate as the metabolite. To account for the detection of PLP and 2 after denaturation, it is suggested that a nonproductive formation of the Schiff base of PLP with 2 occurs in the second subunit of the enzyme; this complex is released and hydrolysed to PLP and 2 upon base denaturation.

4-Aminobutyrate Transaminase↗
PubMed1995

Inactivation of gamma-aminobutyric acid aminotransferase by L-3-chloroalanine hydroxamate.

The mechanism of inactivation of gamma-aminobutyric acid aminotransferase (GABA-AT) by L-3-chloroalanine hydroxamate (1) was investigated. Inactivation of [3H]PLP-reconstituted GABA-AT with 1 followed by denaturation gave no PMP or enamine adduct to the PLP; however, a new unknown metabolite was observed which was identical to the metabolite formed upon inactivation of GABA-AT by L-cycloserine. Time-dependent inactivation occurs, but the kinetics are second order; the rate of inactivation increases with time. After inactivation occurs the addition of fresh enzyme results in a faster rate of inactivation than prior to the initial inactivation. This indicates that the actual inactivator is generated from L-3-chloroalanine hydroxamate, and is not L-3-chloroalanine hydroxamate itself. Added gabaculine-inactivated enzyme to fresh enzyme does not increase the rate of inactivation, suggesting that the conversion of L-3-chloroalanine hydroxamate to the active form is not catalyzed by peripheral amino acid residues. L-3-Chloroalanine hydroxamate was shown to undergo buffer-catalyzed cyclization to L-cycloserine, which is the actual inactivator of GABA-AT.

4-Aminobutyrate Transaminase↗
PubMed1995

Time-dependent inhibition of gamma-aminobutyric acid aminotransferase, by 3-hydroxybenzylhydrazine.

gamma-Aminobutyric acid (GABA) aminotransferase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of GABA into succinic semialdehyde. Hydrazine analogues have long been known to act as inactivators of PLP-dependent enzymes, including GABA aminotransferase, however, no studies of the molecular mechanism of inactivation of PLP-dependent enzymes by hydrazines have been reported. 3-Hydroxybenzylhydrazine is shown to be a potent in vitro time-dependent inhibitor of pig brain GABA aminotransferase. UV-visible and 1H NMR studies, both with GABA aminotransferase and with PLP as a chemical model for the enzyme-catalyzed reaction, indicate that 3-hydroxybenzylhydrazine reacts both enzymatically and nonenzymatically to form the 3-hydroxybenzylhydrazone of PLP without tautomerization.

4-Aminobutyrate Transaminase↗
PubMed1995

SAR studies of fluorine-substituted benzylamines and substituted 2-phenylethylamines as substrates and inactivators of monoamine oxidase B.

To test whether addition of electron-withdrawing substituents to good substrates of monoamine oxidase (MAO) will transform them into inactivators, a series of fluorine-substituted benzylamines and a series of substituted 2-phenylethylamines were synthesized and tested as substrates and inactivators of monoamine oxidase B. All of the compounds were substrates. None of the benzylamines was an inactivator, but several of the phenethylamines were. These results suggest that either stronger electron withdrawing character or additional stabilizing factors are needed to stabilize the hypothesized enzyme adduct.

Animals↗
PubMed1994

Observation of two different chromophores in the resting state of monoamine oxidase B by fluorescence spectroscopy.

Catalytically active monoamine oxidase (MAO) is believed to exist as a dimer with each subunit containing a covalently attached flavin cofactor. Fluorescence spectroscopy performed on the resting state enzyme resulted in two separate fluorescence emissions at 480 nm and 530 nm with excitation maxima at lambda ex = 412 nm and lambda ex = 450 nm, respectively. Inactivation of MAO with pargyline resulted in concomitant loss of the absorbance at 450 nm without change in the 412 nm absorption; there also was a decrease in the emission intensity at 530 nm, while the emission at 480 nm remained unchanged. The 480 nm emission decreased and the 530 nm emission intensity increased, when the enzyme was heat denatured in the presence of NaDodSO4. From these results, it is clear that there are two different chromophores present in the resting state enzyme; the 530 nm chromophore is consistent with an oxidized flavin, while the 480 nm chromophore could be a flavin semiquinone.

Animals↗
PubMed1993

New analogues of N-(2-aminoethyl)-4-chlorobenzamide (Ro 16-6491). Some of the most potent monoamine oxidase-B inactivators.

A series of halo- and nitro-substituted analogues of N-(2-aminoethyl)benzamide has been synthesized. All of the compounds are competitive, time-dependent inhibitors of monoamine oxidase-B (MAO-B), but upon dialysis complete return of enzyme activity is observed for all compounds. Therefore, these are mechanism-based reversible inhibitors of MAO-B. The relative potencies of the compounds are rationalized in terms of steric and hydrophobic effects.

Animals↗
PubMed1993

Transformation of heterocyclic reversible monoamine oxidase-B inactivators into irreversible inactivators by N-methylation.

3-[4-[(3-Chlorophenyl)methoxy]phenyl]-5-[(methylamino)methyl]- 2-oxazolidinone (1) is a secondary amine known to be a potent time-dependent irreversible inactivator of monoamine oxidase B (MAO-B). The primary amine analogues of derivatives of 1, as well as of the corresponding dihydrofuranone and pyrrolidinone, had been shown to be time-dependent, but reversible, inhibitors of MAO-B. Here it is shown that the primary amine analogue of 1 is a time-dependent reversible inhibitor of MAO-B and that the secondary and tertiary amine analogues of the corresponding oxazolidinone, dihydrofuranone, and pyrrolidinone are time-dependent irreversible inhibitors of MAO-B. The reaction leading to the irreversible enzyme adduct formation with 1 can be reversed by increasing the temperature. These results are consistent with a stabilizing stereoelectronic effect on the enzyme adduct caused by N-methylation which hinders free rotation and prevents the sp3-orbital containing the nitrogen nonbonded electrons from being trans to the active site amino acid leaving group.

Animals↗
PubMed1993

The oxazolidinone antibacterial agent DuP 105 does not act on cell wall biosynthesis or on a beta-lactamase.

(S)-3-(4-Methylsulfinylphenyl)-5-(acetamidomethyl)oxazolidinone (DuP 105; 1, R = CH3SO, R' = Ac), one of a new class of antibacterial agents, is shown to be devoid of inactivation and inhibitory properties toward bacterial cell wall biosynthesis and toward a beta-lactamase. This is consistent with the idea that this class of compounds acts by a route different from that of the beta-lactam antibiotics.

Anti-Bacterial Agents↗
PubMed1993

Transformation of monoamine oxidase-B primary amine substrates into time-dependent inhibitors. Tertiary amine homologues of primary amine substrates.

A family of N-methylated and N,N-dimethylated alkyl and arylalkylamines was prepared and more than half of the analogues were shown to be time-dependent pseudo-first-order inhibitors of monoamine oxidase-B. Some of the time-dependent inactivators were reversible and others were irreversible with respect to prolonged dialysis following inactivation. Partition ratios ranged from zero to 11,000. These results are rationalized in terms of a combination of an inductive effect and a stereoelectronic effect as a result of hindered rotation of an active site covalent adduct. A molecular mechanics calculation indicates that there is at least 10 kcal/mol of torsional energy to be overcome in order for the enzyme adduct to be released. These findings show that tertiary amine homologues of primary amine substrates of monoamine oxidase are time-dependent inhibitors, and this should be useful in the design of new inactivators of this enzyme.

Alkylation↗
PubMed1993

Inactivation of monoamine oxidase B by analogues of the anticonvulsant agent milacemide (2-(n-pentylamino)acetamide).

Analogues of the anticonvulsant agent milacemide (1,2-(n-pentylamino)acetamide), in which the carboxamide group is changed to a nitrile (2), a carbethoxy group (3), a carboxylic acid (4), a cyanomethyl group (5), and a trifluoromethyl group (6), were synthesized and tested as substrates and inactivators of monoamine oxidase B (MAO B). The carboxylic acid was neither a substrate nor an inactivator. The trifluoromethyl compound was not soluble in buffer even when organic cosolvents were added, so it could not be tested. All of the other compounds were both substrates and time-dependent irreversible inactivators of MAO B. A plot of the logarithm of kcat/k(inact) (a measure of the efficiency of the inactivators) versus sigma I (Figure 1) shows a linear free energy relationship between the inactivator efficiency and the electron-withdrawing ability of the substituent. As the electron-withdrawing ability increases, the partition ratio decreases indicating that inactivation is becoming more efficient relative to substrate turnover to product. Milacemide was the least efficient of the compounds tested; the nitrile 2 was the most efficient.

Acetamides↗
PubMed1992

3-alkyl GABA and 3-alkylglutamic acid analogues: two new classes of anticonvulsant agents.

Recently we showed that 3-alkyl-4-aminobutanoic acids are in vitro activators of brain L-glutamic acid decarboxylase (GAD) that show anticonvulsant activity. Since activation of GAD leads to increased concentrations of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in vitro, these compounds could represent a new class of anticonvulsant agents. Here it is shown that 3-alkylglutamic acid analogues also activate GAD and that all of the compounds in both series are active anticonvulsant agents against low intensity electroshock in mice. The most active compound, 3-isobutyl GABA, was tested further against maximal electroshock in mice and was shown to be very potent after both intravenous and oral administration without causing ataxia. It is not known if brain GABA levels are elevated in vivo by administration of these compounds or if the mechanism of anticonvulsant activity is related to their ability to activate GAD.

Administration, Oral↗
PubMed1992

Inactivation of gamma-aminobutyric acid aminotransferase by various amine buffers.

It is hypothesized that buffers capable of forming a Schiff base with the PLP of gamma-aminobutyric acid aminotransferase (GABA-AT) may lead to denaturation and inactivation of the enzyme. On the basis of this hypothesis three new methods for the selective destruction of GABA-AT in GABAse (a commercial bacterial source of a mixture of GABA-AT and succinic semialdehyde dehydrogenase [SSDH]) and from pig brain are described: (1) dialysis against a primary or secondary amine buffer; (2) gel filtration with a primary or secondary amine buffer as eluent; (3) inactivation with gabaculine followed by dialysis or gel filtration with pyrophosphate buffer. The SSDH activity in GABAse, which remains unchanged by all of these methods, may then be used in a coupled assay to measure the activity of GABA-AT from different sources. These results also suggest that the use of primary and secondary amine buffers should be avoided when inhibitors are being tested with GABA-AT.

4-Aminobutyrate Transaminase↗