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 spatial structure of cytosolic chicken aspartate aminotransferase (AAT) has been determined by X-ray crystallographic analysis at 2.8 A resolution. AAT consists of two chemically identical subunits. Each subunit can be subdivided into the large pyridoxal phosphate (PLP) binding domain and the small domain. The two active sites of AAT are situated in deep clefts at the subunit interface. The binding of PLP and 2-oxoglutarate is described. Conformations of the following enzyme forms have been compared by difference Fourier syntheses: the nonliganded PLP-form in phosphate and acetate buffers; the non-liganded pyridoxamine phosphate (PMP) form; complexes of the PLP-form with glutarate and 2-oxoglutarate. Lattice-induced dynamic asymmetry of the dimeric AAT molecules was revealed. In one subunit the small domain is mobile and shifted either toward the active site ("closed" conformation) or in the opposite direction ("open" conformation). The closed conformation is induced by the binding of dicarboxylate anions. In the second subunit the small domain is immobile and shifted toward the active site in all enzyme forms or complexes studied. In this subunit, there occurs a rotation of the PLP ring by approximately 20 degrees toward the substrate site. The rotation is observed when crystals are soaked in 0.6 saturated (NH4)2SO4 solution buffered with 0.3 M potassium phosphate, pH 7.5; it was explained by formation of an external aldimine between PLP and NH3. This aldimine is not formed in the presence of dicarboxylates or acetate. It was inferred that dicarboxylate or acetate anions stabilize the internal PLP-lysine aldimine and prevent its reaction with ammonia. Conversion of AAT from the PLP- to PMP-form is accompanied by rotation of the coenzyme ring by approximately 20 degrees; the rotation occurs in both subunits.
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PMID:[Cytosol aspartate aminotransferase from the chicken heart: three-dimensional structure at 2.8 angstroms resolution and the characteristic conformation of various enzyme forms]. 398 8

Cytosolic chicken heart aspartate aminotransferase (EC 2.6.1.1) was incorporated in polyacrylamide gel and partially oriented by compressing the gel block in two mutually perpendicular directions. The linear dichroism (LD) was recorded in a dichrograph equipped with a quarter-wavelength device which transforms circularly polarized light into linearly polarized. Spectra were resolved with lognormal distribution curves. A marked difference has been found between reduced linear dichroism values (LD/A) in the absorption bands of the protonated (430 nm) and nonprotonated (360 nm) forms of the internal pyridoxal phosphate--lysine aldimine. This finding indicates that protonation of the internal aldimine bond induces a change in direction of the transition dipole moment within the coenzyme ring or reorientation of the ring. Formation of the external aldimine with 2-methylaspartate is accompanied by a decrease of the reduced LD value in the 430 nm band. On the other hand, binding of the dicarboxylate anions, which imitates formation of the noncovalent adsorption Michaelis complex, results in a marked increase of the reduced LD value in the 430 nm band. These data suggest that the coenzyme ring tilts in opposite directions upon noncovalent substrate binding and upon subsequent formation of the external aldimine.
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PMID:[Linear dichroism of chicken cytosol aspartate aminotransferase oriented in polyacrylamide gel]. 407 36

1. Acetylation of aspartate aminotransferase from pig heart inhibits completely the enzymic activity when the coenzyme is in the amino form (pyridoxamine phosphate) or when the coenzyme has been removed, but not when the coenzyme is in the aldehyde form (pyridoxal phosphate). 2. The group the acylation of which is responsible for the inhibition has been identified with the in-amino group of a lysine residue at the coenzyme-binding site. Moreover, in the pyridoxamine-enzyme the amino group of the coenzyme is also acetylated. 3. The reactivity of the coenzyme-binding lysine residue is greatly different in the pyridoxamine-enzyme and in the apoenzyme, suggesting the possibility of an interaction of its in-amino group with pyridoxamine or with other groups on the protein.
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PMID:Acylation of aspartate aminotransferase. 604 35

During the transamination reaction of mitochondrial aspartate aminotransferase, transfer of tritium from the alpha-position of glutamate to the pro-S position of C4' of pyridoxamine 5'-phosphate was detected. A fast mixing and quenching device had to be used in order to reduce the number of transamination cycles undergone by the enzyme and thus to minimize the accompanying exchange of label with water. The extent of transfer of label (mean value 1.5%; range 0.8-4%) indicates that the 1,3-prototropic shift follows a stepwise rather than a concerted mechanism and that a single acid/base group is responsible for the proton transfer. The actual extent of proton transfer has to be much higher because the rate of alpha-tritium exchange with solvent was only approximately 10% of that of the turnover of unlabeled substrate, reflecting either an isotope effect or a retention of the tritium label in the reaction center during tautomerization. Under the assumption of an isotope effect, the actual transfer may be estimated to be 13%. This value is consistent with the notion of Lys-258 acting as the proton transferring group in which case the maximal value of transfer in an active site not accessible to solvent during the 1,3-prototropic shift would be 33%. However, alternative mechanisms involving Tyr-70 or a water molecule enclosed in the active site serving as acid/base group cannot be excluded on the basis of the present results. Furthermore, in these investigations aspartate aminotransferase was found to catalyze also the exchange of tritium from the beta-position of glutamate, though at a rate 350 times slower than that of the alpha-exchange.
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PMID:Transfer of C alpha-hydrogen of glutamate to coenzyme of aspartate aminotransferase during transamination reaction. 615 79

Two vitamin B6 derivatives, N-bromoacetylpyridoxamine (BAPM) and its phosphate ester have been found to be affinity-labeling reagents for mitochondrial aspartate aminotransferase (EC 2.6.1.1). These derivatives were first shown to react with a critical sulfhydryl group in tryptophan synthase (Higgins, W., and Miles, E. W. (1978) J. Biol. Chem. 253, 4648-4652). In the apoaminotransferase, BAPM has now been found to inactivate by covalently modifying a critical lysyl residue, preventing reconstitution of the apoenzyme by pyridoxal 5'-phosphate. The dependence of the rate of inactivation upon the concentration of the reagent is consistent with a rapid equilibrium binary complex formation prior to the inactivation reaction. Both the dissociation constant for this complex and the rate of the reaction leading to inactivation are dependent on pH. BAPM binds best from pH 7.5 to 8.5. The rate of inactivation increases from pH 6 to 9. Succinate and phosphate competitively bind to the apoenzyme, protecting against BAPM inactivation. The C-5'-phosphorylated derivative is rapidly and tightly bound by the apotransaminase to form an inactive, noncovalent adduct. This bound reagent subsequently alkylates Lys-258. The rate of this covalent incorporation increases from pH 6 to 9 and is greater than the rate of BAPM modification at all pH values. The effect of pH on the reaction rates of both pyridoxal derivatives is interpreted to indicate protonation of Lys-258 at neutral pH values. These derivatives may also be analogs to a reaction intermediate different from those observed in other affinity-labeling studies. The ionization states of the Lys-258 epsilon-amino group apparently vary with the nature of the affinity label. These variations can be explained in terms of changing ionization states of Lys-258 in the steps of catalysis as well as in terms of the occupancy of charged sites on the protein by active site-directed substrates or inhibitory compounds.
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PMID:Properties of the active site lysyl residue of mitochondrial aspartate aminotransferase in solution. 640 77

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.
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PMID:L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase. 641 46

Experiments were conducted with intact rat hepatocytes to identify inhibitors and incubation conditions that cause selective inhibition of alanine aminotransferase or aspartate aminotransferase. Satisfactory results were obtained by preincubating cells with L-cycloserine or L-2-amino-4-methoxy-trans-but-3-enoic acid in the absence of added substrates. When cells were incubated for 20 min with 50 microM-L-cycloserine, alanine aminotransferase activity was decreased by 90%, whereas aspartate aminotransferase was inhibited by 10% or less. On subsequent incubation, synthesis of glucose and urea from alanine was strongly inhibited, but glucose synthesis from lactate was unaffected. L-2-Amino-4-methoxy-trans-but-3-enoic acid (400 microM) in hepatocyte incubations caused 90-95% inactivation of aspartate aminotransferase, but only 15-30% loss of alanine aminotransferase activity. After preincubation with the inhibitor, glucose synthesis from lactate was almost completely blocked; with alanine as the substrate, gluconeogenesis was unaffected, and urea synthesis was only slightly decreased. By comparison with preincubation with inhibitors, simultaneous addition of substrates (alanine; lactate plus lysine) and inhibitors (cycloserine; aminomethoxybutenoic acid) resulted in smaller decreases in aminotransferase activities and in metabolic rates. Other compounds were less satisfactory as selective inhibitors. Ethylhydrazinoacetate inactivated the two aminotransferases to similar extents. Vinylglycine was almost equally effective in blocking the two enzymes in vitro, but was a very weak inhibitor when used with intact cells. Concentrations of DL-propargylglycine (4 mM) required to cause at least 90% inhibition of alanine aminotransferase in hepatocytes also caused a 16% decrease in aspartate aminotransferase. When tested in vitro, alanine aminotransferase was, as previously reported by others, more sensitive to inhibition by amino-oxyacetate than was aspartate aminotransferase, but in liver cell incubations the latter enzyme was more rapidly inactivated by amino-oxyacetate.
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PMID:Selective inhibition of alanine aminotransferase and aspartate aminotransferase in rat hepatocytes. 646 97

An increase in the HSO3- concentration and in the carbon dioxide level is accompanied by an increase in the glutaminase, alanine and aspartate aminotransferase activities in the liver and kidneys tissues as well as in the blood serum. The in vitro experiments show that an increased level of CO2 in the incubation medium intensifies the incorporation of 14C from I-14C lysine into proteins of the fish liver.
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PMID:[Intensity of metabolism in fish tissues with an increased level of HCO3- and carbon dioxide in their blood]. 677 May 20

The cofactor analogue N-(bromoacetyl)pyridoxamine (BAPM) has been employed to inactivate the cytosolic isozyme of apo-aspartate aminotransferase. Inactivation is the result of covalent bond formation in the (bromoacetyl)-pyridoxamine-transferase complex, via the epsilon-amino group of a lysyl residue at the active site. The stoichiometry of this inactivation is one molecule of (bromoacetyl)pyridoxamine per subunit of the transaminase dimer. Trace amounts of inorganic phosphate protect the enzyme from BAPM inactivation. In the absence of phosphate, inactivation demonstrates time, concentration, and pH dependence with an apparent pK for the target group of about 8.5 or higher. A tryptic peptide from the alpha subform has been obtained containing the carboxymethyl derivative of lysine-258, identifying this particular residue as the reactive group in the region of cofactor binding. Evidence is presented indicating that the pK of Lys-258 appears to be highly dependent upon the electrostatic state of neighboring groups in the active site region. Hence, experimentally obtained values vary according to the chemical nature and charge of the modifying agent or probe.
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PMID:Specific labeling of the active site of cytosolic aspartate aminotransferase through the use of a cofactor analogue, N-(Bromoacetyl)pyridoxamine. 683 38

The interaction between the coenzyme derivative 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate with cytoplasmic and mitochondrial apo-aspartate aminotransferase in the crystalline state was investigated to establish whether the structural differences, known to exist between the active sites of the two isoenzymes in solution, are maintained in the crystal although they are not apparent from the available crystallographic data. In the crystal, as in solution, both apo-isoenzymes reversibly bind the coenzyme derivative and catalyze a slow cleavage reaction, by which pyridoxal 5'-phosphate is produced and bound to the active-site lysine. In the case of the cytoplasmic isoenzyme, however, in the crystal as in solution, the initial complex can follow an alternative reaction path that leads to the formation of a covalent bond between the active-site lysine and the C-5 of the 2,4-dinitrophenyl moiety of the reagent. Therefore, crystal-packing forces neither abolish the active site properties that are needed to cleave the specifically bound reagent and are common to the two isoenzymes nor mask the subtle differences that allow for the selective irreversible labeling of the cytoplasmic isoenzyme.
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PMID:Interaction of a coenzyme analog with aspartate aminotransferase isoenzymes in the crystal. 685 34


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