Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.6.1.1 (aspartate aminotransferase)
21,665 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of ingesting a glucose polymer solution (GP) or water (W) on plasma pyridoxal 5'-phosphate (PLP) and pyridoxal (PL) concentrations were compared in six men (age: 30 +/- 2 y; VO2max: 57.4 +/- 3.2 mL.kg-1.min-1) under running (R) and control (C) conditions. Subjects ran for 2 h at 60-65% of VO2max for R and remained standing for C. For both R and C, 200 mL W or GP was ingested before (0-time) and every 30 min while running (30, 60, and 90 min). Plasma PLP decreased to 95% and 87% of 0-time at 180 min for WC and GPC and increased to 126% and 119% at 90 min and to 124% and 119% at 120 min for WR and GPR. By 60 min postrun, plasma PLP was 98% (WR) and 101% (GPR) of 0-time. There were no significant differences between W and GP conditions. Changes in PLP were not related to plasma volume or blood glucose, free fatty acids, lactate, alkaline phosphatase, aspartate aminotransferase, or alanine aminotransferase. No significant changes in plasma PL were noted. Exercise induces an increase in plasma PLP, perhaps due to transfer of B-6 vitamers from liver to skeletal muscle.
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PMID:Plasma pyridoxal and pyridoxal 5'-phosphate concentrations in response to ingestion of water or glucose polymer during a 2-h run. 198 51

Tyrosine-225 is hydrogen-bonded to the 3'-hydroxyl group of pyridoxal 5'-phosphate in the active site of aspartate aminotransferase. Replacement of this residue with phenylalanine (Y225F) results in a shift in the acidic limb of the pKa of the kcat/KAsp vs pH profile from 7.1 (wild-type) to 8.4 (mutant). The change in the kinetic pKa is mirrored by a similar shift in the spectrophotometrically determined pKa of the protonated internal aldimine. Thus, a major role of tyrosine-225 is to provide a hydrogen bond that stabilizes the reactive unprotonated form of the internal aldimine in the neutral pH range. The Km value for L-aspartate and the dissociation constant for alpha-methyl-DL-aspartate are respectively 20- and 37-fold lower in the mutant than in the wild-type enzyme, while the dissociation constant for maleate is much less perturbed. These results are interpreted in terms of competition between the Tyr225 hydroxyl group and the substrate or quasi-substrate amino group for the coenzyme. The value of kcat in Y225F is 450-fold less than the corresponding rate constant in wild type. The increased affinity of the mutant enzyme for substrates, combined with the lack of discrimination against deuterium in the C alpha position of L-aspartate in Y225F-catalyzed transamination [Kirsch, J. F., Toney, M. D., & Goldberg, J. M. (1990) in Protein and Pharmaceutical Engineering (Craik, C. S., Fletterick, R., Matthews, C. R., & Wells, J., Eds.) pp 105-118, Wiley-Liss, New York], suggests that the rate-determining step in the mutant is hydrolysis of the ketimine intermediate rather than C alpha-H abstraction which is partially rate-determining in wild type.
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PMID:The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km. 198 27

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with [4'-13C]pyridoxal 5'-phosphate (pyridoxal-P). The 13C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4' of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4' was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pKa of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pKa of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4' signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. The intermediate chemical shift of the external aldimine complex is thought to reflect the observation of an equilibrium mixture composed of roughly equal populations of the protonated ketoenamine and a dipolar anion species, corresponding to their respective spectral bands at 430 and 360-370 nm. Conversion to the pyridoxamine form was accomplished via reaction of the internal aldimine with L-cysteinesulfinate or by reduction with sodium borohydride, and the resulting C4' chemical shifts were identified by difference spectroscopy. Finally, the line widths of the C4' resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.
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PMID:Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy. 200 79

In aspartate aminotransferase (AspAT), His143 is located within a hydrogen-bonding distance to Asp222 that forms a strong ion pair with the ring nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). His143 of Escherichia coli AspAT was replaced by Ala or Asn. The mutant enzyme H143A showed a slight increase in the maximum velocity of the overall transamination reaction between aspartate and 2-oxoglutarate, while H143N AspAT showed a decrease to 60% in the maximum rate of the overall reactions in both directions. In all of the half-transamination reactions with four substrates, aspartate, glutamate, oxalacetate, and 2-oxoglutarate, the catalytic competence as defined by kmax/Kd decreased by 3-18-fold upon replacing His143 by either Ala or Asn. The extent of the decrease varied from one substrate to another; it was largely contributed to by the decrease in affinities for all substrates. The equilibrium constants, [PMP-form] [keto acid]/[( PLP-form] [amino acid]), decreased by over 10-fold upon the mutations at position 143. Both H143A and H143N AspATs exhibited a considerably decreased affinity for 2-methylaspartate, an external-aldimine-forming substrate analogue, yet without appreciable alteration in the affinity for succinate and glutarate, which are non-aldimine-forming analogues. All these findings suggest that, although His143 is not essential for catalysis, it might assist the formation of enzyme-substrate complex.
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PMID:The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase. 200 66

Tryptophanase (tryptophan: indole-lyase) from Escherichia coli has been isolated in the holoenzyme form and its absorption spectra and acid-base chemistry have been reevaluated. Apoenzyme has been prepared by dialysis against sodium phosphate and L-alanine and molar absorptivities of the coenzyme bands have been estimated by readdition of pyridoxal 5'-phosphate. The spectrophotometric titration curve, whose midpoint is at pH 7.6 in 0.1 M potassium phosphate buffers, indicates some degree of cooperativity in dissociation of a pair of protons. Resolution of the computed spectra of individual ionic forms of the enzyme with lognormal distribution curves shows that band shapes are similar to those of model Schiff bases and of aspartate aminotransferase. Using molar areas from the latter we estimated amounts of individual tautomeric species. In addition to ketoenamine and enolimine or covalent adduct the high pH form also appears to contain approximately 18% of a species with a dipolar ionic ring (protonated on the ring nitrogen and with phenolate -O-). We suggest that this may be the catalytically active form of the coenzyme in tryptophanase. The equilibrium between tryptophanase and L-alanine has also been reevaluated.
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PMID:Equilibria and absorption spectra of tryptophanase. 203 39

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.
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PMID:Complete amino acid sequence of rat liver cytosolic alanine aminotransferase. 204 42

The vitamin B-6 requirements of 12 men and women over 60 y old were studied. The protocol consisted of a 5-d baseline period and four experimental periods during which the subjects successively received 0.003, 0.015, 0.0225 and 0.03375 mg of vitamin B-6/(kg body wt.d). Dietary protein was 1.2 or 0.8 g/(kg body wt.d). At 5- or 6-d intervals, xanthurenic acid (XA) after a 5-g L-tryptophan load and 4-pyridoxic acid (4-PA) in 24-h urine, erythrocyte aspartate aminotransferase activity coefficient (EAST-AC) and plasma pyridoxal-5'-phosphate (PLP) were measured. These measurements were abnormal during vitamin B-6 depletion but returned to normal during repletion. Men who ingested approximately 120 g protein/d required 1.96 +/- 0.11 mg of vitamin B-6 to normalize XA; women who ingested 78 g protein/d required 1.90 +/- 0.18 mg of vitamin B-6 to normalize XA. To attain normal levels of EAST-AC and 4-PA in men, 2.88 +/- 0.17 mg of vitamin B-6 were needed; to normalize PLP, 1.96 +/- 0.11 mg of vitamin B-6 were required. Women required 1.90 +/- 0.18 mg or more of vitamin B-6 to normalize these measurements. Vitamin B-6 requirements were not decreased in two of three subjects who ingested 54 g of protein daily. Thus, vitamin B-6 requirements of elderly men and women are about 1.96 and 1.90 mg/d, respectively.
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PMID:Vitamin B-6 requirements of elderly men and women. 205 Dec 26

Forty-five male Lohmann chicks were grown up to 6 weeks of age. The experimental diet containing a high protein level (30%) was aimed at increasing the metabolic need for PN. Microbiological analysis on the basal ration revealed a marginal content of 4.7 mumol PN/kg. The vitamin B6 status was assessed at the end of the experiment according to the basal activity of aspartate aminotransferase (AspAT) in plasma and in erythrocytes, and the in vitro stimulated activity with pyridoxal 5'-phosphate (PLP). None of the deficient chicks had any clinical signs attributable to malfunction of the nervous system, and they grew as well as those receiving the control diet. Vitamin B6 deficiency was biochemically confirmed by a significant depression of AspAT activity in plasma (p less than 0.001) and in erythrocytes (p less than 0.01). The addition of PLP in vitro enhanced the catalytic activity of the plasma enzyme, but had negligible effect on the erythrocyte enzyme. The degree of stimulation in vitro of the apoenzyme of AspAT not only depends on the endogenous vitamin B6 content, but also on the basal activity of the enzyme. A 15-day repletion period with a daily oral dose (50 mumol PN) did not result in a complete restoration of the enzyme activity, indicating that the availability of apoenzyme had been curtailed. This experiment demonstrated that chicks fed a high protein corn-soyamin diet with a limited amount of PN but containing Saccharomyces yeast showed no nervous signs or perosis, but significant metabolic disturbances.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Aspartate aminotransferase activity in experimentally induced asymptomatic vitamin B6 deficiency in chicks. 205 99

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm). The rate of this half-reaction of transamination (kcat = 4.0 x 10(-4)s-1) is five orders of magnitude slower than that of the wild-type enzyme. However, the reverse half-reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild-type enzyme, kmax of the observable rate-limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 x 10(-2)s-1. Aspartate aminotransferase (Lys258----His) thus represents a pyridoxal-5'-phosphate-dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction, and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine-ketimine tautomerization.
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PMID:Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence. 210 17

The erythrocyte aspartate aminotransferase and renal and intestinal glycogen phosphorylase activities in rats are determined as dependent on their provision with vitamin B6. It has been shown that the aspartate aminotransferase activity decreases and the shape of the aspartate concentration-activity curve changes in the vitamin B6-deficient animals. The B6 insufficiency does not affect the intestinal mucosa glycogen phosphorylase. However the renal phosphorylase activity decreases by 30 percent in the vitamin B6 deficient rats. It occurs due to changes in the affinity of phosphorylase A and B to glucose-1-phosphate but not to AMP. The activation of these investigated enzymes by exogenous pyridoxal phosphate reveals no essential differences between the vitamin B6-deficient and normal rats. The possible causes of the observed changes in the aspartate aminotransferase and phosphorylase activity are discussed.
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PMID:[Changes in kinetic properties of pyridoxal-dependent enzymes during dietary vitamin B6 deficiency in rats]. 211 Jun 92


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