Abstact

In this study, we report the crystal structures of K214Q and K216Q variants of Escherichia coli glucokinase (ecGLK), each of which is bound to phosphate in the active-site cleft. The structure of the K214Q variant was determined at 2.70 A resolution and refined with an R(work) and R(free) of 0.140 and 0.190, respectively, while that of the K216Q variant was determined at 2.44 A resolution with an R(work) and R(free) of 0.178 and 0.225, respectively. Both variants adopt an open conformation and maintain phosphate-binding interactions similar to the wild-type ecGLK. Structural comparison of the K214Q variant revealed large backbone deviations in the 214-224 alpha-helix, increased disorder in the loops surrounding the glucose-binding cleft and outward shifts of Asn99, Asp100, His160 and Glu187. Our previous study demonstrated that lysine acetylation at Lys214 and Lys216 impaired the activity of ecGLK, and here we show that acetylation mimics produced domain shifts, indicating those of lysine residues that could be essential for stabilizing the glucose-binding region of ecGLK.