Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.     

Phenylalanyl-tRNA synthetase from E. coli MRE-600. Effect of chemical modification of lysine residues on the enzyme interaction with substrates

The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.     

Strain relief at the active site of phosphoserine aminotransferase induced by radiation damage

The X-ray susceptibility of the lysine-pyridoxal-5'-phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 A resolution, complemented by on-line spectroscopic studies. X-rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal-5'-phosphate and the Lys residue. Analysis of the "undamaged" structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X-rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 x 10(6) Ggamma. Our data provide new insights into the enzymatic activation of pyridoxal-5'-phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.     

Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5'-phosphate

An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.     

Immunoblot detection of pyridoxal phosphate binding proteins in liver and hepatoma cytosolic extracts

A monoclonal antibody, highly selective for the 5'-phosphopyridoxyl group, can be used to detect cytosolic pyridoxal-5'-phosphate binding proteins by an immunoblot procedure. This technique, when applied to sodium borohydride-treated cytosolic extracts obtained from normal rat liver at various stages of development as well as several liver-derived Morris hepatomas, reveals patterns of pyridoxal-5'-phosphate binding proteins that are characteristic of the various sources of cytosol. These findings suggest that there are developmental and tumor-specific requirements for pyridoxal-5'-phosphate, the coenzymatically active form of vitamin B-6.     

Aspartate aminotransferase from wheat germ: purification and kinetic properties.

A convenient method for the purification of aspartate aminotransferase [L-aspartate-2-oxoglutarate aminotransferase (EC 2.6.1.1)] from wheat germ is described. An overall purification of 150 fold was achieved. On polyacrylamide gel electrophoresis at pH 8.9 the purified enzyme revealed two protein bands both provided with enzymatic activity. The holoenzyme is readily resolved on conversion to the aminic form and gel-filtration. The apoenzyme is reactivated by pyridoxal-5-phosphate. Kinetic data indicate that a Ping-Pong mechanism is operative similar to that found for the tyrosine aminotransferase by Litwack and Cleland (1968). Phosphate ion behaves as a competitive inhibitor towards the coenzyme. The relatively low affinity between coenzyme and apoenzyme from wheat germ allowed the determination of the dissociation constants for coenzymes (pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate) and of the inhibition constant for phosphate.     

Correlation between nuclear glucocorticoid receptor levels and casein gene expression in murine mammary gland in vitro

The relationship between nuclear binding of glucocorticoid-receptor complex and casein gene expression was studied in organ culture of the whole mammary gland of the mouse. Pyridoxal 5'-phosphate was used as a modulatory agent for measuring nuclear binding of the receptor complex. Addition of 2 mM and 5mM pyridoxal-5'-P in the medium (Waymouth's MB752/1) resulted in 4- and 12-fold increase of its concentration in the glands incubated with insulin, prolactin, and hydrocortisone. Pyridoxal-5'-P also caused a 52% and 92% inhibition of nuclear binding of [3H]dexamethasone in the glands at 2 mM and 5 mM concentration in the presence of the same hormones in the medium. Corresponding to the reduced nuclear binding of the receptor complex casein mRNA levels, measured by a specific cDNA probe was reduced 86% and over 90% in the glands exposed to 2 mM and 5 mM pyridoxal-5'-P, respectively, in presence of insulin, prolactin, and hydrocortisone in the medium. Withdrawal of pyridoxal-5'-P from the medium restored nuclear binding of the receptor complex near the level of control glands incubated only with the hormones. mRNA casein levels also increased in the gland in the pyridoxal-5'-P-free medium containing the same hormones. This indicates that pyridoxal-5'-P does not alter the specific hormone responsiveness of the mammary cells and its action mediated at the level of the glucocorticoid receptor can influence hormone-inducible expression of the casein genes. Thus, glucocorticoid plays a major role in the multiple hormone regulation of the milk protein gene(s). The findings also suggest that the breast tissue concentration of the vitamin B6 derivative may influence the physiology of lactation in nursing mothers.     

Complexes of vitamin B6XX: equilibrium and mechanistic studies of the reaction of pyridoxal-5'-phosphate with pyridoxamine-5'-phosphate in the presence of copper(II)

The interaction of Cu(II) with pyridoxamine-5'-phosphate (PMP) and pyridoxal-5'-phosphate (PLP) was studied potentiometrically. The titration data were assessed by MINIQUAD program. Several protonated and nonprotonated complexes have been found to exist in solution. The reaction of PLP with Cu(II)-PMP has been studied kinetically, using the stopped-flow technique. Two rate steps have been observed. The first step has been attributed to the formation of a Schiff's base metal complex. The second step may be due to the formation of a ternary complex formation. A mechanism was suggested.     

An amine: hydroxyacetone aminotransferase from <Emphasis Type="Italic">Moraxella lacunata</Emphasis> WZ34 for alaninol synthesis

An amine:hydroxyacetone aminotransferase from an isolated soil bacterium, Moraxella lacunata WZ34, was employed to synthesize alaninol in the presence of hydroxyacetone and isopropylamine in this study. The optimal carbon and nitrogen sources were glycerol and beef extract, respectively. A wide range of amino donor specificity was detected with the aminotransferase, which exhibited a relative high activity (9.83 U mL(-1)) in the presence of isopropylamine. The enzyme was the most active at pH 8.5, and showed relatively higher activity at alkaline than acidic pH. Maximum activity was achieved at 30 degrees C, and the enzyme had good thermal stability below 60 degrees C. Metal ions such as Mg(2+) had positive effect (132.6%) on the enzyme, and (aminooxy)acetic acid, a typical aminotransferase inhibitor, significantly inhibited its activity. The enzyme activity was enhanced by the addition of 0.05 mM pyridoxal-5'-phosphate (PLP).     

The role of transaminases in realizing the protective effect of L-aspartate in hypoxia]

Activation of aspartate aminotransferase and alanine aminotransferase of mitochondria introduced to the incubation medium of pyridoxal-5'-phosphate (40 microM) is approximately 2 times higher than that of the corresponding cytoplasmic forms. At hypoxia aspartate aminotransferase activity in mitochondria and postmitochondrial supernatant tends to an increase while that of alanine aminotransferase decreases (above 2 times). The protection from hypoxic damage when using L-aspartate (100 mg/kg subcutaneously 3-5 min before hypoxia) intensifies an adaptive increase of aspartate aminotransferase activity and removes a decrease of alanine aminotransferase activity. Under these conditions stimulating effect of pyridoxal-5'-phosphate on transaminases activity in vitro weakens. A simultaneous administration of vitamin-coenzyme complex (thiamine pyrophosphate, lipoate, sodium 4-phospho-pantothenate, flavin-mononucleotide, nicotinate) intensifies these metabolic shifts and protective action of L-aspartate.     

His230 of serine hydroxymethyltransferase facilitates the proton abstraction step in catalysis.

The three-dimensional structures of rabbit and human liver cytosolic serine hydroxymethyltransferase revealed that H231 interacts with the O3' of pyridoxal-5'-phosphate and other residues at the active site such as S203, K257, H357 and R402 (numbering as per the human enzyme). This and the conserved nature of H231 in all serine hydroxymethyltransferases highlights its importance in catalysis and/or maintenance of oligomeric structure of the enzyme. In an attempt to decipher the role of H230 (H231 of the human enzyme) in the catalytic mechanism and/or maintenance of oligomeric structure of sheep liver serine hydroxymethyltransferase, the residue was mutated to arginine, phenylalanine, alanine, asparagine or tyrosine. Our results suggest that the nature of the amino acid substitution has a marked effect on the catalytic activity of the enzyme. H230R and H230F mutant proteins were completely inactive, dimeric and did not bind pyridoxal-5'-phosphate. On the other hand, mutation to alanine and asparagine retained the oligomeric structure and ability to bind pyridoxal-5'-phosphate. These mutants had only 2-3% catalytic activity. The side reactions like transamination and 5,6,7, 8-tetrahydrofolate independent aldol cleavage were much more severely affected. They were able to form the external aldimine with glycine and serine but the quinonoid intermediate was not observed upon the addition of 5,6,7,8-tetrahydrofolate. Mutation to tyrosine did not affect the oligomeric structure and pyridoxal-5'-phosphate binding. The H230Y enzyme was 10% active and showed a correspondingly lower amount of quinonoid intermediate. The kcat / Km values for L-serine and Lallothreonine were 10-fold and 174-fold less for this mutant enzyme compared to the wild-type protein. These results suggest that H230 is involved in the step prior to the formation of the quinonoid intermediate, possibly in orienting the pyridine ring of the cofactor, in order to facilitate effective proton abstraction.     

Affinity labeling of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active-site cysteine

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 degrees C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-Ala-Cys-Thr-Glu-Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.     

Determination of pyridoxal phosphate levels in the brains of audiogenic and normal mice

The concentrations of pyridoxal-5-phosphate in the brains of audiogenic and normal mice were measured fluorimetrically. The brain of the audiogenic mouse (DBA/2J) contains 25% more pyridoxal-5-phosphate than the brain of a control mouse. Intraperitoneal injection of this substance causes a transient increase of its concentration in the brain, lasting a few hours. The substance thereafter is degraded to pyridoxal and pyridoxic acid.     

X-ray structure refinement and comparison of three forms of mitochondrial aspartate aminotransferase.

The X-ray crystal structures of three forms of the enzyme aspartate aminotransferase (EC 2.6.1.1) from chicken heart mitochondria have been refined by least-squares methods: holoenzyme with the co-factor pyridoxal-5'-phosphate bound at pH 7.5 (1.9 A resolution), holoenzyme with pyridoxal-5'-phosphate bound at pH 5.1 (2.3 A resolution) and holoenzyme with the co-factor pyridoxamine-5'-phosphate bound at pH 7.5 (2.2 A resolution). The crystallographic agreement factors [formula: see text] for the structures are 0.166, 0.130 and 0.131, respectively, for all data in the resolution range from 10.0 A to the limit of diffraction for each structure. The secondary, super-secondary and domain structures of the pyridoxal-phosphate holoenzyme at pH 7.5 are described in detail. The surface area of the interface between the monomer subunits of this dimeric alpha 2 protein is unusually large, indicating a very stable dimer. This is consistent with biochemical data. Both subunit and domain interfaces are relatively smooth compared with other proteins. The interactions of the protein with its co-factor are described and compared among the three structures. Observed changes in co-factor conformation may be related to spectral changes and the energetics of the catalytic reaction. Small but significant adjustments of the protein to changes in co-factor conformation are seen. These adjustments may be accommodated by small rigid-body shifts of secondary structural elements, and by packing defects in the protein core.     

Crystal structures of 1-aminocyclopropane-1-carboxylate (ACC) synthase in complex with aminoethoxyvinylglycine and pyridoxal-5'-phosphate provide new insight into catalytic mechanisms.

The structures of tomato 1-aminocyclopropane-1-carboxylate synthase (ACS) in complex with either cofactor pyridoxal-5'-phosphate (PLP) or both PLP and inhibitor aminoethoxyvinylglycine have been determined by x-ray crystallography. The structures showed good conservation of the catalytic residues, suggesting a similar catalytic mechanism for ACS and other PLP-dependent enzymes. However, the proximity of Tyr152 to the C-gamma-S bond of model substrate S-adenosylmethionine implies its critical role in the catalysis. The concerted accomplishment of catalysis by cofactor PLP and a protein residue, as proposed on the basis of the ACS structures in this paper, may represent a general scheme for the diversity of PLP-dependent catalyses. PLP-dependent enzymes have been categorized into four types of folds. A structural comparison revealed that a core fragment of ACS in fold type I is superimposable over tryptophan synthase beta subunit in fold type II and mouse ornithine decarboxylase in fold type III, thus suggesting a divergent evolution of PLP-dependent enzymes.     

Structural study reveals that ser-354 determines substrate specificity on human histidine decarboxylase

Histamine is an important chemical mediator for a wide variety of physiological reactions. l-Histidine decarboxylase (HDC) is the primary enzyme responsible for histamine synthesis and produces histamine from histidine in a one-step reaction. In this study, we determined the crystal structure of human HDC (hHDC) complexed with the inhibitor histidine methyl ester. This structure shows the detailed features of the pyridoxal-5'-phosphate inhibitor adduct (external aldimine) at the active site of HDC. Moreover, a comparison of the structures of hHDC and aromatic l-amino acid (l-DOPA) decarboxylase showed that Ser-354 was a key residue for substrate specificity. The S354G mutation at the active site enlarged the size of the hHDC substrate-binding pocket and resulted in a decreased affinity for histidine, but an acquired ability to bind and act on l-DOPA as a substrate. These data provide insight into the molecular basis of substrate recognition among the group II pyridoxal-5'-phosphate-dependent decarboxylases.     

Simultaneous and direct determination of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in serum by derivative synchronous fluorescence spectroscopy

Simultaneous and direct assays of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in human serum are described. The method applied is based on the reaction of these compounds with beryllium in an ammoniacal medium to yield highly fluorescent derivatives. Overlapping of conventional fluorescence spectra is resolved by using second-derivative fluorescence spectroscopy, thus making the use of separation techniques unnecessary. The proposed method is simple (only beryllium and an ammoniacal buffer are needed to develop fluorescence), rapid (the derivative formation is instantaneous and serum treatment only requires deproteinization), and inexpensive (no sophisticated detection equipment is necessary, any conventional modern spectrofluorimeter being adequate for use). The analytical recovery achieved was of about 96% for pyridoxal, 97% for pyridoxal-5'-phosphate, and 100% for pyridoxic acid. Measurements were carried out in a single scan.     

Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a bifunctional reagent during the inactivation of porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The biphasic kinetics and X-azolidine-like structure formed were similar to those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo, T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715). In the cytoplasmic enzyme, however, irreversible inactivation representing X-azolidine formation was found to be the dominant characteristic of the interaction with pyridoxal-5'-P. Spectral evidence indicated that at total inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or one pyridoxal-5'-P per enzymatic active site. The presence of NADH protected the enzyme from inactivation suggesting interaction of pyridoxal-5'-P at or near the enzymatic active centers of this enzyme. Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme failed to bind NADH or at least failed to bind NADH in the same fashion as native enzyme.     

Quorum sensing in metal tolerance of Acinetobacter junii BB1A is associated with biofilm production

Acinetobacter junii strain BB1A, a novel metal-tolerant bacterium, produced biofilm in the presence of added ions such as Ni(2+), AsO(2)(-), Cd(2+) and Hg(2+) on surfaces such as glass and polystyrene. Generation of a metal-sensitive and adhesion-deficient mutant by transposition of Tn5-mob in the A. junii genome has putatively confirmed the association of metal tolerance with the production of biofilm. The requirement of a critical cell density for biofilm formation and presence of acyl-homoserine lactone-like autoinducer molecules in the cell-free supernatant indicated the phenomenon of quorum sensing. Addition of a natural quorum-sensing inhibitor (garlic extract) or synthetic quorum-sensing inhibitor (4-nitro-pyridine oxide) significantly inhibited cell growth and biofilm formation in the presence of metal/metalloid ions.     

Alteration of Amino Acid Metabolism in Epileptogenic Mice by Elevation of Brain Pyridoxal Phosphate

A single intraperitoneal injection of pyridoxal-5'-phosphate (PLP) in a species of mouse, DBA/2J, that is normally susceptible to sound-induced convulsion exacerbated its epileptic condition. The effect of injection was most pronounced about 30 min after the administration and subsided gradually within the following 4 h. Correlated with this increased seizure susceptibility were enhanced levels of synaptosomal aspartate and glutamate, and a diminished gamma-aminobutyric acid (GABA) level. The concentrations of nonneuroactive amino acids remained unchanged. When stimulated with veratrine, synaptosomes prepared from PLP-injected mice showed an increased release of aspartate and glutamate and a decreased release of GABA compared to those prepared from control mice. The activity of glutamate decarboxylase in the brains of PLP-treated mice was lowered, whereas the activity of GABA-transaminase was enhanced. Finally, the epileptic condition of DBA mice could be ameliorated by maintenance on a diet composed of vitamin B6-deficient feed and cellulose.