Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.: In vitro intragenic complementation:: The roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization

被引:21
作者
Brokx, SJ
Talbot, J
Georges, F
Waygood, EB
机构
[1] Univ Saskatchewan, Dept Biochem, Saskatoon, SK S7N 5E5, Canada
[2] Natl Res Council Canada, Inst Plant Biotechnol, Saskatoon, SK S7N 0W9, Canada
关键词
D O I
10.1021/bi991250z
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
Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvute:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SE 1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K-m for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V-max of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V-max to 4 and 2%, respectively, and for both, the PEP K-m is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V-max for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K-m for PEP decreased to < 10 mu M, but the K-m became dependent upon the stability of the heterodimer in the assay. Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified, it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain.
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页码:3624 / 3635
页数:12
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