PDBsum entry 1akc

Transferase(aminotransferase)

PDB id: 1akc

Contents

Protein chain

401 a.a. *

Ligands

PPE

Waters ×311

* Residue conservation analysis

References listed in PDB file

Key reference

• Title: Structural basis for the catalytic activity of aspartate aminotransferase k258h lacking the pyridoxal 5'-Phosphate-Binding lysine residue.
• Authors: V.N.Malashkevich, J.Jäger, M.Ziak, U.Sauder, H.Gehring, P.Christen, J.N.Jansonius.
• Ref.: Biochemistry, 1995, 34, 405-414. [DOI no: 10.1021/bi00002a004]
• PubMed id: 7819232

Abstract

Chicken mitochondrial and Escherichia coli aspartate aminotransferases K258H, in which the active site lysine residue has been exchanged for a histidine residue, retain partial catalytic competence [Ziak et al. (1993) Eur. J. Biochem. 211, 475-484]. Mutant PLP and PMP holoenzymes and the complexes of the latter (E. coli enzyme) with sulfate and 2-oxoglutarate, as well as complexes of the mitochondrial apoenzyme with N-(5'-phosphopyridoxyl)-L-aspartate or N-(5'-phosphopyridoxyl)-L-glutamate, were crystallized and analyzed by means of X-ray crystallography in order to examine how the side chain of histidine 258 can substitute as a general acid/base catalyst of the aldimine-ketimine tautomerization in enzymic transamination. The structures have been solved and refined at resolutions between 2.1 and 2.8 A. Both the closed and the open conformations, identical to those of the wild-type enzyme, were observed, indicating that the mutant enzymes of both species exhibit the same conformational flexibility as the wild-type enzymes, although in AspAT K258H the equilibrium is somewhat shifted toward the open conformation. The replacement of the active site K258 by a histidine residue resulted only in local structural adaptations necessary to accommodate the imidazole ring. The catalytic competence of the mutant enzyme, which in the forward half-reaction is 0.1% of that of the wild-type enzyme, suggests that the imidazole group is involved in the aldimine-ketimine tautomerization. However, the imidazole ring of H258 is too far away from C alpha and C4' of the coenzyme-substrate adduct for direct proton transfer, suggesting that the 1,3-prototropic shift is mediated by a water molecule. Although there is enough space for a water molecule in this area, it has not been detected. Dynamic fluctuations of the protein matrix might transiently open a channel, giving a water molecule fleeting access to the active site.

Secondary reference #1

• Title: Mutant aspartate aminotransferase (k258h) without pyridoxal-5'-Phosphate-Binding lysine residue. Structural and catalytic properties.
• Authors: M.Ziak, J.Jäger, V.N.Malashkevich, H.Gehring, R.Jaussi, J.N.Jansonius, P.Christen.
• Ref.: Eur J Biochem, 1993, 211, 475-484.
• PubMed id: 8436109

Secondary reference #2

• Title: Structural basis for catalysis by aspartate aminotransferase
• Authors: J.N.Jansonius, M.G.Vincent.
• Ref.: biological macromolecules, 1987, 3, 188.