Crystal Structures of Escherichia coli Glycerol Kinase Variant S58→W in Complex with Nonhydrolyzable ATP Analogues Reveal a Putative Active Conformation of the Enzyme as a Result of Domain Motion^,

Abstract

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.

Structural and Kinetic Analyses of Arginine Residues in the Active Site of the Acetate Kinase from Methanosarcina thermophila

“…Although in close proximity, neither Arg 241 or His 180 was within strict bonding distance to AlF 3 . However, in a true transition state for an S N 2 reaction, the plane of AlF 3 is perpendicular to the plane connecting the substrate and product (42,(52)(53)(54) 384 is located in the ␣ 3Ј helix of the C-terminal "body" domain, whereas Thr 182 is located in the ␣ 3 helix in the Nterminal "wing" domain (7). Hydrogen bonding has been shown for the corresponding ␣ 3 helices in other ASKHA family members, where it facilitates the domain closure necessary to prevent ATP hydrolysis and promote phosphoryl transfer (43,55,56).…”

Transplanting allosteric control of enzyme activity by protein–protein interactions: Coupling a regulatory site to the conserved catalytic core

“…Positions 427-429 are critical for the binding of IIA Glc and coupling to the active site. Crystal structures show that these amino acids do not form interactions with IIA Glc , and there is no change in conformation in this region after IIA Glc binding that is discernible at the 2.4-3.0-Å resolutions of the structures (7,10,11). No atoms of the amino acids at 427-429 are closer than 4.3 Å to IIA Glc , and no IIA Glc amino acid side chains form polar interactions with these amino acids.…”

“…Crystallographic studies show that the conformations of EcGK residues 474-478 undergo induced-fit transitions between coil, 3 10 helix, and ␣-helix upon IIA Glc binding (11). Alanine substitutions at positions 479-481, which are ␣-helical in the absence or presence of IIA Glc , result in large decreases in affinity that are consistent with stabilization of the ␣-helix, affecting positions that undergo the conformational changes (29).…”

“…Posttranslational regulation of the function of many of the family members is believed to involve modulation of this motion. Crystallographic studies of EcGK show evidence for conformational changes associated with active site closure and induced fit of amino acids that bind IIA Glc but do not show how these two conformational changes are coupled (10,11). The conformation of IIA Glc does not change after binding to EcGK.…”

Orientation of 1,3-Bisphosphoglycerate Analogs Bound to Phosphoglycerate Kinase

“…4 and 5 for a ligand containing only methanephosphonates and a ligand containing both a methanephosphonate and an ␣,␣-difluoromethanephosphonate, respectively. Differential broadening exists of the two 31 P signals, which indicates a conformational preference of the ligand. Line widths were measured by line fitting, and the gradients of line width versus CrADP were compared to derive an effective distance ratio of the two phosphorus atoms from the chromium, as in Equation 5.…”