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Query: UNIPROT:P17174 (aspartate aminotransferase)
 14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN14CO and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NH2-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 +/- 0.08 and k = 146 +/- 5 M-1S-1, which are similar to the values determined for carbamylation of the NH2- terminal groups of human hemoglobin (Garner, M. H., Bogardt, R. A., and Gurd, E. R. N. (1975) J. Biol. Chem. 250, 4398-4404). The pK value for Lys-258 is as low as that for a group in the active site region which can perturb a 19F nuclear magnetic resonance probe inserted into that region (Martinez-Carrion, M., Slebe, J. C., Boettcher, B., and Relimpio, A. M. (1976) J. Biol. Chem. 251, 1853-1858). Apoenzyme carbamylated at Lys-258 can accept pyridoxal phosphate at the active site even though no Schiff base in formed. Furthermore, this active site carbamylated holoenzyme will form spectroscopically detectable enzyme-substrate complexes with amino acids. The complexes slowly convert to species with absorbance identical with that of enzyme in the pyridoxamine phosphate form.
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PMID:Carbamylation of aspartate transaminase and the pK value of the active site lysyl residue. 96 83

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.
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PMID:Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus. 155 94

We have carried out a Fourier transform infrared spectroscopic study of mitochondrial aspartate aminotransferase in the spectral region where phosphate monoesters give rise to absorption. Infrared spectra in the above-mentioned region are dominated by protein absorption. Yet, below 1020 cm-1 protein interferences are minor, permitting the detection of the band arising from the symmetric stretching of dianionic phosphate monoesters [T. Shimanouchi, M. Tsuboi, and Y. Kyogoku (1964) Adv. Chem. Phys. 8, 435-498]. The integrated intensity of this band in several enzyme forms (pyridoxal phosphate, pyridoxamine phosphate, and sodium borohydride-reduced, pyridoxyl phosphate form) does not change with pH in the range 5-9. This behavior contrasts that of free pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) in solution, where the dependence of the same infrared band intensity with pH can be correlated to the known pK values for the 5'-phosphate ester in solution. The integrated intensity value of this infrared band for the PLP enzyme form before and after reduction with sodium borohydride is close to that given by free PLP at pH 8-9. These results are taken as evidence that in the active site of mitochondrial aspartate aminotransferase the 5'-phosphate group of PLP remains mostly dianionic even at a pH near 5. Thus, it is suggested that the chemical shift changes associated with pH titrations of various PLP forms reported in a previous 31P NMR study of this enzyme [M. E. Mattingly, J. R. Mattingly, and M. Martinez-Carrion (1982) J. Biol. Chem. 257, 8872] are due to the fact that the phosphorus chemical shift senses the O-P-O bond distortions induced by the ionization of a nearby residue. Since no chemical shift changes were observed in pH titrations of the PMP forms (lacking an ionizable internal aldimine) of this isozyme, the Schiff base between PLP and Lys-258 at the active site is the most likely candidate for the ionizing group influencing the phosphorus chemical shift in this enzyme.
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PMID:The ionization states of the 5'-phosphate group in the various coenzyme forms bound to mitochondrial aspartate aminotransferase. 189 57

The precursor to rat liver mitochondrial aspartate aminotransferase has been expressed in Escherichia coli JM105 using the pKK233-2 expression vector. This mammalian natural precursor has been isolated as a soluble dimeric protein. The amino-terminal sequence and the amino acid composition of the isolated protein correspond to those predicted from the inserted cDNA (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). The isolated precursor contains bound pyridoxal phosphate and shows catalytic activity with a specific activity equal to that of the mature form of the enzyme. This precursor can also be processed by mitochondria into a form with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of mature enzyme. The isolation of this precursor as a stable and catalytically active entity indicates that the presequence peptide does not necessarily interfere with much of the folding and basic structural properties of the mature protein component.
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PMID:Isolation and properties of a liver mitochondrial precursor protein to aspartate aminotransferase expressed in Escherichia coli. 264 43

Cytosolic and mitochondrial aspartate aminotransferase cDNAs were cloned from a lambda gt11 rat liver cDNA library. The complete coding sequence and the 3' non-coding sequence of the cytosolic isozyme mRNA were obtained from two overlapping cDNA clones. Partial sequences of the mitochondrial enzyme cDNAs were found to be identical to the recently published complete sequence (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). A single mRNA (2.4 kb (kilobase pair] hybridizing to the mitochondrial cDNA probe was detected by Northern blot analysis, whereas the cytosolic cDNA probe labeled one major (2.1 kb) and two minor (1.8 and 4 kb) mRNAs. The 1.8-kb and the 2.1-kb cytosolic aspartate aminotransferase mRNAs differ in their 3' ends and probably result from the use of either of the two polyadenylation signals present in the 3' noncoding region of the major cytosolic aspartate aminotransferase mRNA. Glucocorticoid hormones increased the activity of cytosolic but not mitochondrial aspartate aminotransferase in both liver and kidney. The increase in the enzyme activity was accompanied by an increase in the amount of the three corresponding mRNAs, while the mitochondrial enzyme mRNA was not significantly modified.
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PMID:Nucleotide sequence and glucocorticoid regulation of the mRNAs for the isoenzymes of rat aspartate aminotransferase. 318 56

The enzyme, aspartate aminotransferase, is a dimer consisting of two identical subunits which contain overlapping subunit regions ( Eichele , G., Ford, G.C., Glor , M., Jansonius , J.N., Mavrides , C., and Christen , P. (1979) J. Mol. Biol. 133, 161-180), suggesting the possibility of subunit interactions. The structurally similar cytosolic isozyme exhibits noncooperative binding of pyridoxal 5'-phosphate ( Boettcher , M., and Martinez -Carrion, M. (1975) Biochemistry 14, 4528-4531; Relimpio , A., Iriarte , A., Chlebowski , J.F., and Martinez -Carrion, M. (1981) J. Biol. Chem. 256, 4478-4488) in which the apoenzyme/holoenzyme hybrid dimer shows a distinctive thermal stability. Using a nonequilibrium isoelectric focusing technique, it can be shown that mitochondrial aspartate aminotransferase also binds cofactor in a noncooperative random fashion. However, differential scanning calorimetry (DSC) thermograms show different characteristics from the cytosolic form. These differences are interpreted in terms of unique subunit interactions in this isozyme. Heating to the various DSC transition temperatures shows that the anomalous DSC thermograms in partially coenzyme-saturated apoenzyme preparations are due to a selective dissociation of apoenzyme subunits into monomers which are irreversibly denatured. The remaining holoenzyme monomers reassociate and form stable holoenzyme dimers. The net result is retention of the initial concentration of holoenzyme subunits present in any given mixture. Random occupancy of active sites and similar electrophoretic and DSC patterns upon heating of partially saturated apoenzyme preparations is observed whether the coenzyme, pyridoxal phosphate or pyridoxamine phosphate alone, or borohydride-reduced Schiff's bases of coenzyme-substrate analogue derivatives are used as active site directed ligands. The latter resemble covalent enzyme-substrate intermediates.
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PMID:Coenzyme active site occupancy as an indicator of independence of the subunits of mitochondrial aspartate aminotransferase. 672 80

Differential scanning calorimetry has been applied to study factors affecting the thermally induced denaturation of cytoplasmic aspartate aminotransferase, a dimeric pyridoxal enzyme. The consequences of binding of coenzyme and substrate derivatives to both the apo and holo forms of the enzyme were investigated and are interpreted in terms of the stabilization of the native form of the enzyme. The binding of pyridoxal phosphate coenzyme increases the thermal stability of the apoenzyme by approximately 27 kcal mol-1 as judged by the change in free energy differences between the native and denatured states of the protein. The stabilization produced by coenzyme binding to the apoprotein appears to be primarily due to the Schiff's base and phosphoryl moieties of the coenzyme; association of the pyridine ring component is without significant structural consequence. Pyridoxal phosphate binding to the subunits of the dimer occurs in a noncooperative fashion as judged by the appearance of transitions unique to the apo, holo, and intermediate enzyme forms in a calorimetric titration. Holoenzyme stability depends on the chemical nature of the catalytically significant group occupying the C-4' position of the bound coenzyme. The stabilization afforded by binding of the aldehyde form (pyridoxal phosphate) which exists as an internal Schiff's base with Lys 258 is diminished when this bond is chemically reduced or when the aldehyde is replaced by an amine (pyridoxamine phosphate). Apoenzyme is also shown to be stabilized by the presence of substrates in the absence of coenzyme. The differential scanning calorimetry results thus confirm previous findings derived from nuclear magnetic resonance studies on the ability of apoenzyme to bind substrates (Martinez-Carrion, M. Cheng, S., and Relimpio, A. (1973) J. Biol. Chem. 248, 2153-2160). Substrates and their analogues perturb the holoenzyme stability and the order of increasing influence on the pyridoxal form of the holoenzyme is aspartate, erythro-hydroxyaspartate, alpha-ketoglutarate, and alpha-methylaspartate. While all these compounds form stable binary enzyme-substrate complexes (Jenkins, W.T., and D'Ari, L. (1966) J. Biol. Chem. 541, 5667-5674), the complex with alpha-methylaspartate produces anomalous changes in the protein structure which are reflected in the calorimetric parameters. This suggests that caution be exercised in the use of analogues as substrate substitutes in crystallographic work. Differential scanning calorimetry also appears as a sensitive method with which to study the stereochemical dependence of ligand binding on enzyme-induced thermal stabilization. This is illustrated by the use of 4-carbon dicarboxylic acids where only those in the conformation favorable for binding are effective in stabilizing the holoenzyme.
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PMID:Differential scanning calorimetry of cytoplasmic aspartate transaminase. 721 92

The precursor (pmAspAT) and mature (mAspAT) forms of mitochondrial aspartate aminotransferase interact with hsp70 very early during translation when synthesized in either rabbit reticulocyte lysate or wheat germ extract (Lain, B., Iriarte, A., and Martinez-Carrion. (1994) J. Biol. Chem. 269, 15588-15596). The nature of the structural elements responsible for recognition and binding of this protein to hsp70 has been studied by examining the folding and potential association with the chaperone of several engineered forms of this enzyme. Whereas pmAspAT and mAspAT bind hsp70 very early during translation, the cytosolic form of this enzyme (cAspAT) does not interact with hsp70. A fusion protein consisting of the mitochondrial presequence peptide attached to the amino terminus of cAspAT associates with hsp70 only after the protein has acquired its native-like conformation, apparently through binding to the presequence exposed on the surface of the folded protein. Deletion of the amino-terminal segment of mAspAT or its replacement with the corresponding domain from the cytosolic isozyme eliminates the cotranslational binding of hsp70 to the mitochondrial protein. We conclude that both the presequence and NH2-terminal region of pmAspAT represent recognition signals for binding of hsp70 to the newly synthesized mitochondrial precursor. Results from competition studies with synthetic peptides support this conclusion. The ability of hsp70 to discriminate between these two highly homologous proteins probably involves the recognition of specific sequence elements in the NH2-terminal portion of the mitochondrial protein and may relate to their separate localization in the cell. A slower folding rate and higher affinity for cytosolic chaperones may represent evolutionary adaptations of translocated mitochondrial proteins to ensure their efficient importation into the organelle.
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PMID:Structural features of the precursor to mitochondrial aspartate aminotransferase responsible for binding to hsp70. 755 89

The homologous cytosolic and mitochondrial isozymes of aspartate aminotransferase (c- and mAspAT, respectively) seem to follow very different folding pathways after synthesis in rabbit reticulocyte lysate, suggesting that the nascent proteins interact differently with molecular chaperones (Mattingly, J. R., Jr., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 26320-26327). In an attempt to discern the structural basis for this phenomenon, we have begun to study the effect of temperature on the refolding of the guanidine hydrochloride-denatured, purified proteins and their interaction with the groEL/groES molecular chaperone system from Escherichia coli. In the absence of chaperones, temperature has a critical effect on the refolding of the two isozymes, with mAspAT being more susceptible than cAspAT to diminishing refolding yields at increasing temperatures. No refolding is observed for mAspAT at physiological temperatures. The molecular chaperones groEL and groES can extend the temperature range over which the AspAT isozymes successfully refold; however, cAspAT can still refold at higher temperatures than mAspAT. In the absence of groES and MgATP, the two isozymes interact differently with groEL, groEL arrests the refolding of mAspAT throughout the temperature range of 0-45 degrees C. Adding only MgATP releases very little mAspAT from groEL; both groES and MgATP are required for significant refolding of mAspAT in the presence of groEL. On the other hand, the extent to which groEL inhibits the refolding of cAspAT depends upon the temperature of the refolding reaction, only slowing the reaction at 0 degrees C but arresting it completely at 30 degrees C. MgATP alone is sufficient to effect the release of cAspAT from groEL at any temperature examined; inclusion of groES along with MgATP has no effect on the refolding yield but does increase the refolding rate at temperatures greater than 15 degrees C. These results demonstrate that groEL can have significantly different affinities for proteins with highly homologous final tertiary and quarternary structures and suggest that dissimilarities in the primary sequence of the protein substrates may control the structure of the folding intermediates captured by groEL and/or the composition of the surfaces through which the folding proteins interact with groEL.
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PMID:Homologous proteins with different affinities for groEL. The refolding of the aspartate aminotransferase isozymes at varying temperatures. 783 72

The mitochondrial isozyme of aspartate aminotransferase (mAspAT), a dimeric pyridoxal phosphate (PLP)-dependent enzyme, is encoded by the nuclear genome and synthesized in the cytoplasm as a precursor protein (pmAspAT) containing a 29-residue amino-terminal signal peptide which is essential for its targeting and import into mitochondria. In the cytosolic-like environment of rabbit reticulocyte lysate, newly synthesized rat liver pmAspAT has been found to slowly fold and bind PLP (Mattingly, J. R., Jr., Youssef, J., Iriarte, A. and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). On the other hand, isolated mammalian (pig) mAspAT, when denatured with guanidine hydrochloride, seems unable to refold to a catalytically active state (West, S. M., and Price, N. C. (1990) Biochem. J. 265, 45-50). With the availability of rat liver recombinant precursor and mature forms of mAspAT as homogeneous, stable preparations, an assessment of the influence of the signal peptide on the in vitro refolding of this protein can be made. Following unfolding induced by guanidine hydrochloride, we have investigated the refolding process of this complex, dimeric coenzyme-dependent protein system by activity, fluorescence, and circular dichroism. Both mAspAT and pmAspAT can be efficiently renatured after rapid dilution of the denaturing agent at low protein concentrations. The equilibrium unfolding/refolding transitions and the kinetics of folding are protein concentration-independent and identical for both protein forms. Binding of coenzyme into the active site pocket seems to occur at a late step in the folding process of both mAspAT and pmAspAT, suggesting that in these proteins the coenzyme does not direct the folding of the polypeptide chain. These results indicate that the in vitro refolding of mAspAT is not regulated or influenced by the presence of the amino-terminal signal peptide. On the other hand, in vitro refolding in buffer is significantly faster than the folding of newly synthesized precursor protein in reticulocyte lysate examined in our previous report (reference above), pointing at the likely influence of cytosolic factors in modulating folding in the cell.
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PMID:Refolding of the precursor and mature forms of mitochondrial aspartate aminotransferase after guanidine hydrochloride denaturation. 822 37


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