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

被引:51
作者
Bystrom, CE
Pettigrew, DW
Branchaud, BP
O'Brien, P
Remington, SJ [1 ]
机构
[1] Univ Oregon, Inst Mol Biol, Dept Phys, Eugene, OR 97403 USA
[2] Univ Oregon, Dept Chem, Eugene, OR 97403 USA
[3] Texas A&M Univ, Dept Biochem & Biophys, College Stn, TX 77483 USA
关键词
D O I
10.1021/bi982460z
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 IIA(Glc); 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 similar to 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.
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页码:3508 / 3518
页数:11
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