Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P17174 (aspartate aminotransferase)
 14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pyridoxamine 5'-phosphate in 18 microliters of human capillary blood plasma is determined by catalytic amplification using the apoenzyme of aspartate aminotransferase. Prior isolation from interfering substances is accomplished by employment of a cation exchange resin in batch operation. The procedure consists of the following stages. Stage I, denaturation of proteins. Trichloroacetic acid is used to precipitate plasma proteins and liberate any bound coenzyme. Dilute NaCl is added to expand the volume thus minimizing coenzyme entrapment in the precipitate. Stage II, isolation of the coenzyme. A sulfonated polystyrene ion exchange resin is used inside a centrifugal filter. Pyridoxamine 5'-phosphate in the supernatant from Stage I adsorbs to the resin. Pyridoxal 5'-phosphate, other organic phosphates, and Pi are removed by centrifugation. Rinsing with dilute NaBH4 destroys traces of pyridoxal 5'-phosphate and washes off residual inhibitors. Pyridoxamine 5'-phosphate is then desorbed with NaOH and Tris buffer and recovered by centrifugation. Stage III, reconstitution and assay. The desorbate from Stage II is incubated with excess apoenzyme. Specific activity of the reconstituted enzyme is measured. Interpolation from a standard curve relating enzyme specific activity and pyridoxamine 5'-phosphate concentration yields the plasma level of the cofactor. Approximately 3 h are required to carry out the procedure. Much of the coenzyme was found not be assayable if plasma was refrigerated overnight or if whole blood was left standing at room temperature for a few hours. The degradation was arrested with freezing at -80 degrees C. In a 13-day experiment involving a healthy subject, sharp rises of plasma pyridoxamine 5'-phosphate were found to occur in response to small doses of oral vitamin B6.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Determination of pyridoxamine 5'-phosphate in human blood plasma. 180 57

In an attempt to change the reaction and substrate specificity of aspartate aminotransferase, several apolar active-site residues were substituted in turn with a histidine residue. Aspartate aminotransferase W140H (of Escherichia coli) racemizes alanine seven times faster (Kcat' = 2.2 x 10(-4) s-1) than the wild-type enzyme, while the aminotransferase activity toward L-alanine was sixfold decreased. X-ray crystallographic analysis showed that the structural changes brought about by the mutation are limited to the immediate environment of H140. In contrast to the tryptophan side chain in the wild-type structure, the imidazole ring of H140 does not form a stacking interaction with the coenzyme pyridine ring. The angle between the two ring planes is about 50 degrees. Pyridoxamine 5'-phosphate dissociates 50 times more rapidly from the W140H mutant than from the wild-type enzyme. A model of the structure of the quinonoid enzyme substrate intermediate indicates that H140 might assist in the reprotonation of C alpha of the amino acid substrate from the re side of the deprotonated coenzyme-substrate adduct in competition with si-side reprotonation by K258. In aspartate aminotransferase I17H (of chicken mitochondria), the substituted residue also lies on the re side of the coenzyme. This mutant enzyme slowly decarboxylates L-aspartate to L-alanine (Kcat' = 8 x 10(-5) s-1). No beta-decarboxylase activity is detectable in the wild-type enzyme. In aspartate aminotransferase V37H (of chicken mitochondria), the mutated residue lies besides the coenzyme in the plane of the pyridine ring; no change in reaction specificity was observed. All three mutations, i.e. W140-->H, I17-->H and V37--H, decreased the aminotransferase activity toward aromatic amino acids by 10-100-fold, while decreasing the activity toward dicarboxylic substrates only moderately to 20%, 20% and 60% of the activity of the wild-type enzymes, respectively. In all three mutant enzymes, the decrease in aspartate aminotransferase activity at pH values lower than 6.5 was more pronounced than in the wild-type enzyme, apparently due to the protonation of the newly introduced histidine residues. The study shows that substitutions of single active-site residues may result in altered reaction and substrate specificities of pyridoxal-5'-phosphate-dependent enzymes.
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PMID:Substitution of apolar residues in the active site of aspartate aminotransferase by histidine. Effects on reaction and substrate specificity. 785 26