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)

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple ACC synthase was overexpressed in Escherichia coli (3 mg/liter) and purified to near homogeneity. A continuous assay was developed by coupling the ACC synthase reaction to the deamination of 5'-methylthioadenosine by adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Aspergillus oryzae. The enzyme is dimeric, with kcat = 9s-1 per monomer and Km = 12 microM for AdoMet. The pyridoxal phosphate-binding site of ACC synthase appears to be highly homologous to that of aspartate aminotransferase, suggesting similar roles for corresponding residues. Site-directed mutagenesis of Lys-273, Arg-407, and Tyr-233 (corresponding to residues 258, 386, and 225 in aspartate aminotransferase) and kinetic analyses of the mutants confirms their importance in the ACC synthase mechanism. The Lys-273 to Ala mutant has no detectable activity, supporting the identification of this residue as the base catalyzing C alpha proton abstraction. Mutation of Arg-407 to Lys results in a precipitous drop in kcat/Km and an increase in Km for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate alpha-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the Km for AdoMet and no change in kcat, suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.
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PMID:Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms. 780 54

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway. ACS shares the conservation of 11 invariant residues with a family of aminotransferases that includes aspartate aminotransferase. Site-directed mutagenesis on two of these residues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly reduces enzymatic activity, indicating their importance in catalysis. These mutants have been used in complementation experiments either in vivo in Escherichia coli or in an in vitro transcription/translation assay to study whether the enzyme functions as a dimer. When the Y92L mutant is coexpressed with the K278A mutant protein, there is partial restoration of enzyme activity, suggesting that the mutant proteins can dimerize and form active heterodimers. Coexpressing a double mutant with the wild-type protein reduces wild-type activity, indicating that inactive heterodimers are formed between the wild-type and the double mutant protein subunits. Furthermore, hybrid complementation shows that another tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-ACS4 have limited capacity for heterodimerization. The data suggest that ACS functions as a dimer with shared active sites.
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PMID:Complementation analysis of mutants of 1-aminocyclopropane- 1-carboxylate synthase reveals the enzyme is a dimer with shared active sites. 957 9

The 2.4 A crystal structure of the vitamin B6-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase is described. This enzyme catalyses the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. ACC synthase has 15 % sequence identity with the well-studied aspartate aminotransferase, and a completely different catalytic activity yet the overall folds and the active sites are very similar. The new structure together with available biochemical data enables a comparative mechanistic analysis that largely explains the catalytic roles of the conserved and non-conserved active site residues. An external aldimine reaction intermediate (external aldimine with ACC, i.e. with the product) has been modeled. The new structure provides a basis for the rational design of inhibitors with broad agricultural applications.
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PMID:Structure of 1-aminocyclopropane-1-carboxylate synthase, a key enzyme in the biosynthesis of the plant hormone ethylene. 1061 Jul 93

The active sites of the homologous pyridoxal phosphate- (PLP-) dependent enzymes 1-aminocyclopropane-1-carboxylate (ACC) synthase and aspartate aminotransferase (AATase) are almost entirely conserved, yet the pK(a)'s of the two internal aldimines are 9.3 and 7.0, respectively, to complement the substrate pK(a)'s (S-adenosylmethionine pK(a) = 7.8 and aspartate pK(a) = 9.9). This complementation is required for maximum enzymatic activity in the physiological pH range. The most prominent structural difference in the active site is that Ile232 of ACC synthase is replaced by alanine in AATase. The I232A mutation was introduced into ACC synthase with a resulting 1.1 unit decrease (from 9.3 to 8.2) in the aldimine pK(a), thus identifying Ile232 as a major determinant of the high pK(a) of ACC synthase. The mutation also resulted in reduced k(cat) (0.5 vs 11 s(-1)) and k(cat)/K(m) values (5.0 x 10(4) vs 1.2 x 10(6) M(-1) s(-1)). The effect of the mutation is interpreted as the result of shortening of the Tyr233-PLP hydrogen bond. Addition of the Y233F mutation to the I232A ACC synthase to generate the double mutant I232A/Y233F raised the pK(a) from 8.2 to 8.8, because the Y233F mutation eliminates the hydrogen bond between that residue and PLP. The introduction of the retro mutation A224I into AATase raised the aldimine pK(a) of that enzyme from 6.96 to 7.16 and resulted in a decrease in single-turnover k(max) (108 vs 900 s(-1) for aspartate) and k(max)/K(m)(app) (7.5 x 10(4) vs 3.8 x 10(5) M(-1) s(-1)) values. The distance from the pyridine nitrogen of the cofactor to a conserved aspartate residue is 2.6 A in AATase and 3.8 A in ACC synthase. The D230E mutation introduced into ACC synthase to close this distance increases the aldimine pK(a) from 9.3 to 10.0, as would be predicted from a shortened hydrogen bond.
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PMID:Modulation of the internal aldimine pK(a)'s of 1-aminocyclopropane-1-carboxylate synthase and aspartate aminotransferase by specific active site residues. 1188 3

The three-dimensional structure of the pyridoxal 5'-phosphate (PLP)-dependent L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica is described here. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. The molecule is a molecular dimer where each subunit consists of a large and small domain. Overall the protein is very similar to the members of the family of aspartate aminotransferases. Indeed, the arrangement of the ligands surrounding the cofactor and putative substrate binding site are remarkably close to that observed in histidinol phosphate aminotransferase, which suggests that this latter enzyme might have been its progenitor. The only significant differences in structure occur at the N-terminus, which is approximately 12 residues shorter in CobD and does not form the same type of interdomain interaction common to other aminotransferases. CobD is unusual since within the aspartate aminotransferase subfamily of PLP-dependent enzymes the chemical transformations are substantially conserved, where the only exceptions are 1-aminocyclopropane-1-carboxylate synthase and CobD. Although there are a large number of PLP-dependent amino acid decarboxylases, these are generally larger and structurally distinct from the members of the aspartate aminotransferase subfamily of enzymes. The structure of CobD suggests that the chemical fate of the external aldimine can be redirected by modifications at the N-terminus of the protein. This study provides insight into the evolutionary history of the cobalamin biosynthetic pathway and raises the question of why most PLP-dependent decarboxylases are considerably larger enzymes.
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PMID:Three-dimensional structure of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica. 1193 74