PDBsum entry: 1ari

Transferase (aminotransferase)

PDB id: 1ari

Contents

Protein chains

396 a.a. *

Ligands

PLP ×2

MAE ×2

Waters ×707

* Residue conservation analysis

PDB id:

1ari

Name:

Transferase (aminotransferase)

Title:

Aspartate aminotransferase, w140h mutant, maleate complex

Structure:

Aspartate aminotransferase. Chain: a, b. Synonym: aspartate transaminase. Engineered: yes. Mutation: yes. Other_details: racemizes alanine seven times faster than wi aspartate aminotransferase

Source:

Escherichia coli. Organism_taxid: 562. Strain: ty103. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.30Å R-factor: 0.210

Authors:

V.N.Malashkevich,J.N.Jansonius

Key ref:

R.A.Vacca et al. (1995). Substitution of apolar residues in the active site of aspartate aminotransferase by histidine. Effects on reaction and substrate specificity. Eur J Biochem, 227, 481-487. PubMed id: 7851426 DOI: 10.1111/j.1432-1033.1995.tb20413.x

Date:

23-Aug-95 Release date: 14-Nov-95

Protein chains

P00509  (AAT_ECOLI) -  Aspartate aminotransferase

Seq: 396 a.a.

Struc: 396 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.6.1.1 - Aspartate transaminase.
• Reaction: L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate

L-aspartate (Bound ligand (Het Group name = MAE) matches with 88.89% similarity) + 2-oxoglutarate = oxaloacetate + L-glutamate

• Cofactor: Pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (Bound ligand (Het Group name = PLP) matches with 93.75% similarity)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (2 terms)
• Biological process: biosynthetic process (4 terms)
• Biochemical function: catalytic activity (10 terms)

reference

DOI no: 10.1111/j.1432-1033.1995.tb20413.x

Eur J Biochem 227:481-487 (1995)

PubMed id: 7851426

Substitution of apolar residues in the active site of aspartate aminotransferase by histidine. Effects on reaction and substrate specificity.

R.A.Vacca, P.Christen, V.N.Malashkevich, J.N.Jansonius, E.Sandmeier.

ABSTRACT

In an attempt to change the reaction and substrate specificity of aspartate aminotransferase, several apolar active-site residues were substituted in turn with a histidine residue. Aspartate aminotransferase W140H (of Escherichia coli) racemizes alanine seven times faster (Kcat' = 2.2 x 10(-4) s-1) than the wild-type enzyme, while the aminotransferase activity toward L-alanine was sixfold decreased. X-ray crystallographic analysis showed that the structural changes brought about by the mutation are limited to the immediate environment of H140. In contrast to the tryptophan side chain in the wild-type structure, the imidazole ring of H140 does not form a stacking interaction with the coenzyme pyridine ring. The angle between the two ring planes is about 50 degrees. Pyridoxamine 5'-phosphate dissociates 50 times more rapidly from the W140H mutant than from the wild-type enzyme. A model of the structure of the quinonoid enzyme substrate intermediate indicates that H140 might assist in the reprotonation of C alpha of the amino acid substrate from the re side of the deprotonated coenzyme-substrate adduct in competition with si-side reprotonation by K258. In aspartate aminotransferase I17H (of chicken mitochondria), the substituted residue also lies on the re side of the coenzyme. This mutant enzyme slowly decarboxylates L-aspartate to L-alanine (Kcat' = 8 x 10(-5) s-1). No beta-decarboxylase activity is detectable in the wild-type enzyme. In aspartate aminotransferase V37H (of chicken mitochondria), the mutated residue lies besides the coenzyme in the plane of the pyridine ring; no change in reaction specificity was observed. All three mutations, i.e. W140-->H, I17-->H and V37--H, decreased the aminotransferase activity toward aromatic amino acids by 10-100-fold, while decreasing the activity toward dicarboxylic substrates only moderately to 20%, 20% and 60% of the activity of the wild-type enzymes, respectively. In all three mutant enzymes, the decrease in aspartate aminotransferase activity at pH values lower than 6.5 was more pronounced than in the wild-type enzyme, apparently due to the protonation of the newly introduced histidine residues. The study shows that substitutions of single active-site residues may result in altered reaction and substrate specificities of pyridoxal-5'-phosphate-dependent enzymes.