Changing the Reaction Specificity of a Pyridoxal-5'-phosphate-dependent Enzyme

Graber, Rachel; Kasper, Patrik; Malashkevich, V.N.; Sandmeier, Erika; Berger, Philipp; Gehring, Heinz; Jansonius, Johan N.; Christen, Philipp

doi:10.1111/j.1432-1033.1995.tb20861.x

Cited by 40 publications

(42 citation statements)

References 23 publications

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“…Three remaining changes (Ser-257-Ala, *Arg-289-*Leu and Lys-383-Ser) took place in the regions directly associated with the transamination reaction, closely to highly conservative Lys258 residue which takes part in external aldimine formation with substrate, and two arginins (*Arg292 and Arg386) of which guanidine groups form hydrogen bonds with carboxylate groups of substrate (*Arg292 with a distal group and Arg386 with proximal group) and play a crucial role in ATT substrate binding [11]. Substitution of one of these two arginines, or both of them simultaneously, with amino acids having different physicochemical properties resulted in a decrease of k cat /K m values for at least three or six orders of magnitude, respectively [9,10,[39][40][41]. The occurrence of Ser257, *Arg289 and Lys383 in AAT-2 instead of Ala, *Leu and Ser in AAT-3 should increase the positive electrostatic charge (*Arg289 and Lys383) and the potential to form the hydrogen bonds (Ser257, *Arg289) in the area of arginine (*Arg292 and Arg386) side chains of plastidial allozymes comparing with those from the cytosol.…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

2013

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Six allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k cat) and the k cat/K m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s−1/10,412–10,795 s−1 M−1 for AAT-2 zone and 4.6–7.0 s−1/527–700 s−1 M−1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.

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Section: Discussionmentioning

confidence: 99%

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

2013

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“…During the catalytic cycle, the cofactor shuttles between the PLP and the pyridoxamine 5Ј-phosphate (PMP) forms. High resolution x-ray crystallographic analyses (3)(4)(5)(6) in conjunction with site-directed mutagenesis studies (7)(8)(9)(10)(11)(12)(13)(14) have elucidated the role of several active-site residues. The specificity for dicarboxylic amino acids appears to be based mainly on two active-site arginine residues (Fig.…”

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confidence: 99%

Active-site Arg → Lys Substitutions Alter Reaction and Substrate Specificity of Aspartate Aminotransferase

Vacca¹,

Giannattasio²,

Graber³

et al. 1997

Journal of Biological Chemistry

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and Arg 386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the ␤-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (k cat ‫؍‬ 0.002 s ؊1 ) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg 3 Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.The pyridoxal 5Ј-phosphate (PLP) 1 -dependent enzymes that catalyze transformations of amino acids (for a recent review, see Ref. 1) constitute a few families of evolutionarily related enzymes (2). The member enzymes of such a family use the same protein scaffold to catalyze quite diverse reactions. Apparently, subtle structural differences underlie their catalytic specificity.Aspartate aminotransferase (AspAT) is probably the most extensively studied PLP-containing enzyme. It catalyzes the reversible transamination of the dicarboxylic L-amino acids, aspartate and glutamate, and the corresponding 2-oxo acids, oxalacetate and 2-oxoglutarate. During the catalytic cycle, the cofactor shuttles between the PLP and the pyridoxamine 5Ј-phosphate (PMP) forms. High resolution x-ray crystallographic analyses (3-6) in conjunction with site-directed mutagenesis studies (7-14) have elucidated the role of several active-site residues. The specificity for dicarboxylic amino acids appears to be based mainly on two active-site arginine residues (Fig.

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“…The addition of serine or glycine to rSHMT or P297R caused an appreciable quenching of fluorescence, whereas the addition of -aspartate had no effect, suggesting that the conversion of Pro-297 to Arg had not induced an ability to bind Asp (results not shown). It was therefore not surprising that transamination of -Asp was not observed with the mutant enzyme in a coupled assay system [26]. The effect of mutation on alternative reactions such as H % -folate-independent transamination of -alanine was also examined.…”

Section: Spectral and Catalytic Properties Of The P297r Mutant Enzymementioning

confidence: 99%

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Talwar

Leelavathy

Rao

et al. 2000

Biochemical Journal

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Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5´-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85–90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5´-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with l-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

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Changing the Reaction Specificity of a Pyridoxal-5'-phosphate-dependent Enzyme

Cited by 40 publications

References 23 publications

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Active-site Arg → Lys Substitutions Alter Reaction and Substrate Specificity of Aspartate Aminotransferase

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

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