Crystal Structures of Escherichia coli Aspartate Aminotransferase in Two Conformations

Jäger, Joachim; Moser, Markus; Sauder, Ursula; Jansonius, Johan N.

doi:10.1006/jmbi.1994.1368

Cited by 190 publications

(217 citation statements)

References 0 publications

Supporting

Mentioning

205

Contrasting

Order By: Relevance

“…The approximately 30" forward rotation (in the view of Fig. 3) of the coenzyme is immediately apparent, and is similar to that observed experimentally with aspartate aminotransferase (Jansonius & Vincent, 1987;McPhalen et al, 1992;Jager et al, 1994) and proposed for DGD (Toney et al, 1995). This change in coenzyme orientation is brought about by the formation of an aldimine bond to GABA (displacing Lys 329), by the relief of steric interactions between P L P C4' and Lys 329 NE once this new bond is formed, and by the relaxation of contacts between the Val 300 side chain and the pyridine ring.…”

Section: Discussionsupporting

confidence: 84%

“…A double hydrogen bond-salt bridge between Arg 445 and Glu 270 is also hypothesized; this would provide charge neutralization, as does the a-carboxylate interaction in the glutamate external aldimine. Jansonius et al (1994) proposed an analogous interaction for the ornithine aminotransferase catalyzed reaction.…”

Section: Resultsmentioning

confidence: 99%

“…The 3D-ID profiles (Bowie et al, 1991) for the GABA-AT and DGD models are very similar in form. Models for the obligatory external aldimine intermediates were built based on the structure of the holoenzyme, and on coenzyme rearrangements that have been observed experimentally in aspartate aminotransferase structures (Jansonius & Vincent, 1987;McPhalen et al, 1992;Jager et al, 1994). The complete holoenzyme model and the active site regions of the external aldimine models are available upon request to the authors.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Toney

Pascarella²,

Biase³

1995

Protein Science

View full text Add to dashboard Cite

A homology model for the pig isozyme of the pyridoxal phosphate-dependent enzyme y-aminobutyrate (GABA) aminotransferase has been built based mainly on the structure of dialkylglycine decarboxylase and on a multiple sequence alignment of 28 evolutionarily related enzymes. The proposed active site structure is presented and analyzed. Hypothetical structures for external aldimine intermediates explain several characteristics of the enzyme. In the GABA external aldimine model, the p r o 4 proton at C4 of GABA, which abstracted in the 1,3-azaallylic rearrangement interconverting the aldimine and ketimine intermediates, is oriented perpendicular to the plane of the pyridoxal phosphate ring. Lys 329 is in close proximity and is probably the general base catalyst for the proton transfer reaction. The carboxylate group of GABA interacts with Arg 192 and Lys 203, which determine the specificity of the enzyme for monocarboxylic w-amino acids such as GABA. In the proposed structure for the L-glutamate external aldimine, the a-carboxylate interacts with Arg 445. Glu 265 is proposed to interact with this same arginine in the GABA external aldimine, enabling the enzyme to act on w-amino acids in one half-reaction and on a-amino acids in the other. The reactivities of inhibitors are well explained by the proposed active site structure. The R and S isomers of &substituted phenyl andp-chlorophenyl GABA would bind in very different modes due to differential steric interactions, with the reactive S isomer leaving the orientation of the GABA moiety relatively unperturbed compared to that of the natural substrate. In our model, only the reactive S isomer of the mechanism-based inhibitor vinyl-GABA, an effective anti-epileptic drug known clinically as Vigabatrin, would orient the scissile C4-H bond perpendicular to the coenzyme ring plane and present the proton to Lys 329, the proposed general base catalyst of the reaction. The R isomer would direct the vinyl group toward Lys 329 and the C4-H bond toward Arg 445. The active site model presented provides a basis for site-directed mutagenesis and drug design experiments.Keywords: y-aminobutyrate aminotransferase; GABA; homology modeling; pyridoxal phosphate; Vigabatrin y-Aminobutyrate aminotransferase is a pyridoxal phosphatedependent homodimeric enzyme distributed widely in nature (Cooper, 1985, and references therein). It catalyzes, via a pingpong bi-bi kinetic mechanism, the reversible transfer of the y-amino group of GABA to a-ketoglutarate to yield succinic semialdehyde and L-glutamate, i.e.,

show abstract

Section: Discussionsupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Toney

Pascarella²,

Biase³

1995

Protein Science

View full text Add to dashboard Cite

show abstract

“…The small domain, which is composed of Ct and Nt residues, rotates towards the active site upon substrate or inhibitor binding thus shifting the enzyme from an open into a closed conformation. 37 Our partial non-competitive inhibition data shows that binding of an inhibitor into an active site, that is, locking that active site in the closed conformation, results in a slight decrease in the k cat of the second site. The decrease of k AAT cat for OCT/rWT with respect to SRHEPT þ A293D can therefore be explained by the preferential binding of Asp to the reduced AATase site, which effectively locks that active site into the closed conformation.…”

Section: Putative Model To Explain the Allosteric Communicationmentioning

confidence: 72%

“…It is therefore possible that small movements of the Nt-tail might be able to affect the activity of the opposite subunit. Also, the crystal structures of eAATase 12,37 and our stability studies 14 show that the interface between the two large domains is formed by a large interconnected network of electrostatic interactions and hydrogen bonds. This might mediate the weak allosteric effects observed in this study.…”

Section: Putative Model To Explain the Allosteric Communicationmentioning

confidence: 81%

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

Deu

Kirsch

2011

Protein Science

View full text Add to dashboard Cite

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate-(AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

show abstract

Cysteine-191 in aspartate aminotransferases appears to be conserved due to the lack of a neutral mutation pathway to the functional equivalent, alanine-191

1996

View full text Add to dashboard Cite

Crystal Structures of Escherichia coli Aspartate Aminotransferase in Two Conformations

Cited by 190 publications

References 0 publications

Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Active site model for γ‐aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

Cysteine-191 in aspartate aminotransferases appears to be conserved due to the lack of a neutral mutation pathway to the functional equivalent, alanine-191

Contact Info

Product

Resources

About