Rotational dynamics of 4-aminobutyrate aminotransferase

The fluorescence dye 1-anilinonaphthalene-8-sulfonate (ANS) was used as a probe of non-polar binding sites in 4-aminobutyrate aminotransferase. ANS binds to a single binding site of the dimeric protein with a K_d of 6 μM. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (φ = 65 ns) corresponds to the rotation of a rather rigid dimeric structure. The microenvironment surrounding the natural probe pyridoxal-5-P covalently bound to the dimeric structure was explored using ³¹P-NMR at 72.86 MHz. In the native enzyme, the pyridoxal-5-P ³¹P-chemical shift is pH-independent, indicating that the phosphate group is well protected from the solvent. The correlation time determined from the ³¹P-spectrum of the aminotransferase exceeds the value calculated for the hydrated spherical model (φ = 40 ns). It is concluded that the phosphate of the pyridoxal-5-P molecule is rigidly bound to the active site of 4-aminobutyrate aminotransferase.     

The reaction of ethanolamine O-sulphate with 4-aminobutyrate aminotransferase.

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.     

Kinetics of inactivation of 4-aminobutyrate aminotransferase by 3-bromopyruvate.

3-Bromopyruvate inhibited 4-aminobutyrate aminotransferase (EC 2.6.1.19) from Pseudomonas fluorescens, apparently irreversibly. Kinetics of this inactivation were studied by continuously monitoring the enzyme reaction at 30 degrees C in the presence of inhibitor. Irrespective of how high an inhibitor concentration was present, a maximum rate of inactivation was eventually achieved (5.9 x 10(-3) s-1), indicating the formation of a reversible inhibitor-enzyme complex before the final inactivation step. The dissociation constant of this complex was found to be 6.5 microM. This affinity labelling by 3-bromopyruvate suggests the presence of essential sulphydryl groups on the enzyme, since this compound is known to preferentially alkylate cysteinyl residues.     

Kinetic and spectral properties of rabbit brain 4-aminobutyrate aminotransferase.

Purification and 4-aminobutyrate-2-oxoglutarate aminotransferase (EC 2.6.1.19) from rabbit brain is described. The method was used as a routine to give between 5 and 10mg of pure enzyme from 750 g of rabbit brain. The enzyme is a dimer made up of subunits each with a mol. wt. of 58000. An absorption spectrum of the freshly prepared enzyme shows peaks at 415 and 330 nm. Treatment of the enzyme with the substrate 4-amino-butyrate or glutamate produces a decrease in the 415 nm and an increase in the 330 nm peak. This conversion, which is attributed to an aldimine into ketimine step in the reaction, is sufficiently slow when 4-aminobutyrate is the substrate to allow it to be followed by stopped-flow spectrophotometry. A first-order rate constant was determined for this step (12s-1) and compared with the turnover number for the enzyme derived by steady-state methods (9.5S-1). The first-order rate constant when glutamate was used as substrate was estimated to be approx. 30s-1.     

Catalytic and structural properties of trypsin-treated 4-aminobutyrate aminotransferase

4-Aminobutyrate aminotransferase (EC 2.6.1.19, 4 aminobutyrate:2-oxoglutarate aminotransferase) is cleaved by trypsin, yielding an enzymatically active species which can be separated from the split peptides by gel filtration. The shortened enzyme derivative gives one band (Mr = 95,000) on polyacrylamide gradient gel electrophoresis. Changes in protein conformation induced by tryptic digestion were studied by fluorescence spectroscopy. The native enzyme tagged with the chromophore fluorescein yields a rotational relaxation time of 106 ns, whereas the trypsin-digested enzyme gives a rotational relaxation time of 33 ns. The decrease in rotational relaxation time is attributed to flexibility of the polypeptide chain with enhanced rotational freedom of the probe covalently linked to one thiol group. The reactivity of sulfhydryl groups toward 5,5'-dithiobis(2-nitrobenzoic acid) is also affected by trypsin cleavage. More--SH groups (2.6/dimer) become reactive toward 5,5'-dithiobis(2-nitrobenzoic acid) as a result of trypsin digestion. Local conformational fluctuations are induced as a result of tryptic cleavage, but the catalytic sites remain intact. The peptides released from 4-aminobutyrate aminotransferase were characterized by fingerprint analysis and their amino acid composition determined.     

Mapping of the arginine deiminase gene in Pseudomonas aeruginosa

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.     

Active site modification of 4-aminobutyrate aminotransferase with ATP analogs

The dialdehyde of oxidized 1,N6-etheno-ATP and adenosine triphosphopyridoxal were used as probes of the catalytic site of 4-aminobutyrate aminotransferase. Both compounds react with lysine residues critically connected with aminotransferase activity. The binding of 1 mol of oxidized 1,N6-etheno-ATP per mol of enzyme or the binding of 1 mol of adenosine triphosphopyridoxal abrogates catalytic activity. The presence of substrate alpha-ketoglutarate (4 mM) prevents inactivation of the aminotransferase by either one of the ATP analogs. Reduction of the enzyme modified with oxidized 1,N6-etheno-ATP yields a chromophore which displays a maximum of emission at 415 nm and a fluorescent lifetime of 21.6 ns. The degree of exposure of the ethenoadenine ring to collisional encounters with the strong quencher KI was determined at pH 7.0. The ethenoadenine ring of the bound ligand is partially shielded from collisional encounters with the quencher. Steady-state emission anisotropy measurements of the bound ligand reveal that oxidized 1,N6-etheno-ATP is not rigidly attached to the protein matrix. It is postulated that the catalytic domain of 4-aminobutyrate aminotransferase is accessible to bulky reagents of greater length than the substrates 4-aminobutyrate and alpha-ketoglutarate.     

Brain 4-aminobutyrate aminotransferase. Isolation and sequence of a cDNA encoding the enzyme.

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It is responsible for the conversion of the neurotransmitter 4-aminobutyrate to succinic semialdehyde. By using oligonucleotide probes based on partial amino acid sequence data for the pig brain enzyme, several overlapping cDNA clones of 2.0-2.2 kilobases in length have been isolated. The largest cDNA clone was selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the precursor of 4-aminobutyrate aminotransferase consists of the mature enzyme of 473 amino acid residues and an amino-terminal segment of 27 amino acids attributed to the signal peptide. The cofactor pyridoxal-5-P is bound to lysine residue 330 of the deduced amino acid sequence of the mature enzyme.     

Purification and properties of 4-aminobutyrate 2-ketoglutarate aminotransferase from pig liver.

4-Aminobutyrate-transaminase (4-aminobutyrate: 2-oxoglutarate amino-transferase, EC 2.6.1.19) from pig liver has been purified to electrophoretic homogeneity. It has a molecular weight of about 110 000 and is composed of two subunits of the same molecular weight but of different charges. Two forms of pig liver 4-aminobutyrate-transaminase were isolated by DEAE-cellulose chromatography and designated as 4-aminobutyrate-transaminase I and 4-aminobutyrate-transaminase II, corresponding to a cationic and anionic form. Some physical and kinetic properties of liver enzyme were compared to those of brain enzyme and no significant difference were found, except for their sedimentation coefficients and the charges of their subunits. The role of 4-aminobutyrate-transaminase in liver remains a matter of speculation, but could be related to a metabolic function.     

Regulation of enzyme synthesis in the arginine biosynthetic pathway of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the synthesis of only two out of eight arginine biosynthetic enzymes tested was regulated. Comparisons were made between the specific activities of these enzymes in bacteria grown on arginine or on its precursor, glutamate. N2-Acetylornithine 5-aminotransferase (ACOAT), an enzyme involved in both the biosynthesis and catabolism of arginine, was induced about 14-fold during growth of the organism on arginine as the only carbon and nitrogen source, and the anabolic ornithine carbamoyltransferase (aOTC), a strictly biosynthetic enzyme, was repressed 18-fold. Addition of various carbon sources to the arginine medium led to repression of ACOAT and to derepression of aOTC. Fructose, which supported only slow growth of P. aeruginosa, had a weak regulatory effect on the synthesis of the two arginine enzymes while citrate, a good carbon source for this organism, had a strong effect. The repression of ACOAT by citrate was not relieved by adding cyclic AMP to the medium. Under a variety of growth conditions leading to different enzyme activities, a linear relationship between the reciprocal of the specific activity of ACOAT and the specific activity of aOTC was observed. This inverse regulation of the formation of the two enzymes suggested that a single regulatory system governs their synthesis. Such a view was supported by the isolation of citrate-resistant regulatory mutants which constitutively formed ACOAT at the induced level and aOTC at the repressed level.     

Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa.

The three enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa strain PAO were induced strongly (50- to 100-fold) by a shift from aerobic growth conditions to very low oxygen tension. Arginine in the culture medium was not essential for induction, but increased the maximum enzyme levels twofold. The induction of the three enzymes arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3) appeared to be coordinate. Catabolic ornithine carbamoyltransferase was studied in most detail. Nitrate and nitrite, which can replace oxygen as terminal electron acceptors in P. aeruginosa, partially prevented enzyme induction by low oxygen tension in the wild-type strain, but not in nar (nitrate reductase-negative) mutants. Glucose was found to exert catabolite repression of the deiminase pathway. Generally, conditions of stress, such as depletion of the carbon and energy source or the phosphate source, resulted in induced synthesis of catabolic ornithine carbamoyltransferase. The induction of the deiminase pathway is thought to mobilize intra- and extracellular reserves of arginine, which is used as a source of adenosine 5'-triphosphate in the absence of respiration.     

Role of arginine residues in the active site of the membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis of both sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 from Escherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in the soluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residues were targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined. The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, were shown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays and SUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determined for the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in K(M) values, significant decreases in k(cat) values suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilization of both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 are vital for proper orientation of the substrate in the active site, and furthermore this supports the proposed role of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that would appear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity or activity.     

Co-purification of alanine-glyoxylate aminotransferase with 2-aminobutyrate aminotransferase in rat kidney

Alanine-glyoxylate aminotransferase and 2-aminobutyrate aminotransferase were co-purified from rat kidney to a single protein (about 500-fold purified from the homogenate). The activity ratios of alanine-glyoxylate aminotransferase to 2-aminobutyrate aminotransferase were constant during co-purification steps suggesting the 2-aminobutyrate aminotransferase activity was catalysed by only alanine-glyoxylate aminotransferase. The molecular weight of the enzyme was estimated to be approx. 213 000, 220 000 and 236 000 by analytical ultracentrifugation, Sephadex G-150 gel filtration and sucrose density gradient centrifugation, respectively. From the polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the enzyme consisted of four apparently similar subunits having a molecular weight of approx. 56 000. The enzyme was almost specific to L-alanine and L-2-aminobutyrate as amino donor and to glyoxylate, pyruvate and 2-oxobutyrate as amino acceptor. The enzyme was identified with rat liver alanine-glyoxylate aminotransferase isoenzyme 2 but not with rat liver alanine-glyoxylate aminotransferase isoenzyme 1 from Ouchterlony double diffusion analysis. Absorption spectra and some kinetic properties of the enzyme were clarified.     

Chemistry of the inactivation of 4-aminobutyrate aminotransferase by the antiepileptic drug vigabatrin

The chemical modification of pig liver 4-aminobutyrate aminotransferase by the antiepileptic drug 4-aminohex-5-enoate (Vigabatrin) has been studied. After inactivation by 14C-labeled Vigabatrin, the enzyme was digested with trypsin, and automated Edman degradation of the purified labeled peptide gave the sequence FWAHEHWGLDDPADVMTFSKK. Chymotryptic digestion of the tryptic peptide and sequencing of a resulting tripeptide identified the penultimate lysine residue of this peptide as the site of covalent modification. This lysine normally binds the coenzyme. Absorption spectroscopy demonstrated the absence of coenzyme from the tryptic peptide, and mass spectrometry showed its mass/charge ratio to be increased by 128. All of the bound coenzyme released after denaturation of the inactivated enzyme was as pyridoxamine phosphate. The structural nature of the modification is deduced, and mechanisms for its occurrence identified. Initially, 1 mol of radiolabeled inhibitor was bound per mol of monomer of the enzyme, although approximately half was released during denaturation and digestion, while the remainder was irreversibly bound. Coenzyme not released as pyridoxamine phosphate retained the absorbance characteristics of the aldimine, although the enzyme was completely inactive. Mass spectrometry of the sample of purified radiolabeled tryptic peptide revealed the presence of an approximately equal amount of a second fragment that contained no modification and from which the second lysine was absent, indicating that at the time of proteolysis the active site lysine was unaltered in 50% of the enzyme molecules.     

The mature size of rat 4-aminobutyrate aminotransferase is different in liver and brain.

The amino acid sequence predicted from a rat liver cDNA library indicated that the precursor of beta-AlaAT I (4-aminobutyrate aminotransferase, beta-alanine-oxoglutarate aminotransferase) consists of a mature enzyme of 466 amino acid residues and a 34-amino acid terminal segment, with amino acids attributed to the leader peptide. However, the mass of beta-AlaAT I from rat brain was larger than that from rat liver and kidney, as assessed by Western-blot analysis, mass spectroscopy and N-terminal sequencing. The mature form of beta-AlaAT I from the brain had an ISQAAAK- peptide on the N-terminus of the liver mature beta-AlaAT I. Brain beta-AlaAT I was cleaved to liver beta-AlaAT I when incubated with fresh mitochondrial extract from rat liver. These results imply that mature rat liver beta-AlaAT I is proteolytically cleaved in two steps. The first cleavage of the motif XRX( downward arrow)XS is performed by a mitochondrial processing peptidase, yielding an intermediate-sized protein which is the mature brain beta-AlaAT I. The second cleavage, which generates the mature liver beta-AlaAT I, is also carried out by a mitochondrial endopeptidase. The second peptidase is active in liver but lacking in brain.     

The fourth arginine catabolic pathway of Pseudomonas aeruginosa

D-Arginine dehydrogenase activity was discovered in Pseudomonas aeruginosa. This enzyme was inducible by its substrate, D-arginine, as well as by its product, 2-ketoarginine, but not by L-arginine. The enzyme activity was measured in vitro, in the presence of artificial electron acceptore (phenazine methosulphate and iodonitrotetrazolium chloride). 2-ketoarginine was catabolized further to 4-guanidinobutyraldehyde, 4-guanidinobutyrate and 4-aminobutyrate. Two enzymes involved, 4-guanidinobutyraldehyde dehydrogenase and guanidinobutyrase, were inducible by 2-ketoarginine; the latter enzyme was also strongly induced by 4-guanidinobutyrate. An arginine racemase activity was detected by an invivo test. E-Arginine had the potential to be catabolized via the D-arginine dehydrogenase pathway and, after racemization, via the three L-arginine catabolic pathyways previously demonstrated in P. aeruginosa. In mutants blocked in the L-arginine succinyltransferase pathway, but no in the wild-type, L-arginine was channelled partially into the D-arginine dehydrogenase pathway. Mutations in the kauB locus abolished growth of P. aeruginosa on 2-ketoarginine, agmatine and putrescine, and led to loss of 4-guanidinobutyraldehyde dehydrogenase and 4-aminobutyaldehyde dehydrogenase activites. Thus, these two activites appear to be due to one enzyme in P. aeruginosa. The kauB locus was mapped on the chromosome between lysA and argB and was not linked to known genes involved in the three L-arginine catabolic pathways. The existence of four arginine catabolic pathways illustrates the metabolic versatility of P. aeruginosa.