Biochemical and X-ray crystallographic studies on shikimate kinase: The important structural role of the P-loop lysine

Citation
T. Krell et al., Biochemical and X-ray crystallographic studies on shikimate kinase: The important structural role of the P-loop lysine, PROTEIN SCI, 10(6), 2001, pp. 1137-1149
Citations number
68
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
10
Issue
6
Year of publication
2001
Pages
1137 - 1149
Database
ISI
SICI code
0961-8368(200106)10:6<1137:BAXCSO>2.0.ZU;2-M
Abstract
Shikimate kinase, despite low sequence identity, has been shown to be struc turally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of res idues in the P-loop of shikimate kinase, which forms the binding site for n ucleotides and is one of the most conserved structural features in proteins . In common with many members of the P-loop family, shikimate kinase contai ns a cysteine residue 2 amino acids upstream of the essential lysine residu e; the side chains of these residues are shown to form an ion pair. The C13 S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme . The structure of the K15M mutant protein has been determined at 1.8 Angst rom, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K 15M mutant also reveals that the formation of an additional arginine/aspart ate ion pair is the most likely reason for its increased thermostability. F rom studies of ligand binding it appears that, like adenylate kinase, shiki mate kinase binds substrates randomly and in a synergistic fashion, indicat ing that the two enzymes have similar catalytic mechanisms.