The use of natural and unnatural amino acid substrates to define the substrate specificity differences of<i>escherichia coli</i>aspartate and tyrosine aminotransferases

Onuffer, James; Ton, Binh T.; Klement, Ivan A.; Kirsch, Jack F.

doi:10.1002/pro.5560040909

Cited by 32 publications

(23 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Wild-type AspAT shows an inverse relationship between k cat /K m values and the side chain length of dicarboxylic amino acids (Table I), as has also been shown in another study (32). The side chain of lysine is shorter than that of arginine, the C ␣ -N distance being 5.64 and 6.50 Å, respectively.…”

Section: Discussionsupporting

confidence: 81%

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

Vacca¹,

Giannattasio²,

Graber³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 81%

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

Vacca¹,

Giannattasio²,

Graber³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The preference of eAATase for dicarboxylic amino acid substrates and eTATase for both dicarboxylic and nonpolar amino acids is presented in Onuffer et al (1995). Aspartate and phe- nylalanine were chosen as representative amino acid substrates for monitoring progress in the successive redesign of eAATase, as these two substrates exhibit the highest second-order rate constants in transamination reactions of eAATase and eTATase, respectively.…”

Section: Catalysismentioning

confidence: 99%

“…The single turnover transamination kinetics of aspartate and phenylalanine were followed and analyzed as described in Onuffer et al (1995).…”

Section: Single-turnover Kineticsmentioning

confidence: 99%

Redesign of the substrate specificity ofescherichia coliaspartate aminotransferase to that ofescherichia colityrosine aminotransferase by homology modeling and site-directed mutagenesis

Onuffer

Kirsch

1995

Protein Sci.

Self Cite

View full text Add to dashboard Cite

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in a n attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase.Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes. Examination of the dissociation constants of the dicarboxylate inhibitor maleate and the aromatic inhibitor hydrocinnamate with the mutant constructs demonstrates that the T109S and N297S mutations are specific determinants for high-affinity association of nonpolar ligands, whereas the other four mutations have the general effect of decreasing the dissociation constants for both dicarboxylate and nonpolar ligands. The latter four changes presumably exert their general effect by stabilizing the closed conformation of the enzyme that is observed in X-ray crystal structures of eAATase complexed with dicarboxylate ligands.

show abstract

“…Our molecular docking analysis of the new analogues based on the modeling study which was performed to understand the binding mode of these analogues with the aspartate amino transferase (ATT) of E. coli [43]binding pocket (PDB code: 1ahg, [44]). …”

Section: Molecular Modeling Analysismentioning

confidence: 99%

Synthesis, antimicrobial activity, computational and modeling studies of some new organotellurium compounds containing azo moities

et al. 2015

View full text Add to dashboard Cite

The use of natural and unnatural amino acid substrates to define the substrate specificity differences ofescherichia coliaspartate and tyrosine aminotransferases

Cited by 32 publications

References 35 publications

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

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

Redesign of the substrate specificity ofescherichia coliaspartate aminotransferase to that ofescherichia colityrosine aminotransferase by homology modeling and site-directed mutagenesis

Synthesis, antimicrobial activity, computational and modeling studies of some new organotellurium compounds containing azo moities

Contact Info

Product

Resources

About