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
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.