Kinetic Comparison of the Specificity of the Vancomycin Resistance Kinase VanS for Two Response Regulators, VanR and PhoB
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- 作者：Stewart L. Fisher ; Soo-Ki Kim ; Barry L. Wanner ; and Christopher T. Walsh
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：April 16, 1996
- 年：1996
- 卷：35
- 期：15
- 页码：4732 - 4740
- 全文大小：291K
- 年卷期：v.35,no.15(April 16, 1996)
- ISSN：1520-4995
文摘
Induction of vancomycin resistance in the Gram-positiveEnterococci requires a two-componentregulatory system, VanS and VanR, for transcriptional activation ofthree genes (vanH, A, X) thatencodeenzymes for a cell wall biosynthetic pathway that produces an alteredpeptidoglycan intermediate withlower affinity for the antibiotic. The catalytic efficiency(kcat/KM) has beendetermined for phosphotransferfrom the phosphohistidyl form of VanS to both its homologous partnerVanR and the heterologous(Escherichia coli) response regulator PhoB.The rate of formation of the phosphoaspartyl forms ofVanRand PhoB were determined as well as the rate of appearance of inorganicphosphate. Using PhoB inexcess of P-VanS, a pseudo-first-order rate constant(kxfer) of 0.2 min-1for phosphotransfer and a KM forPhoB of 100 
M were readily determined. The correspondingkxfer of 96 min-1 forphosphotransfer fromP-VanS to VanR required rapid quench kinetics. AKM of 3 M was estimated for VanR, leading toa104-fold preference inkxfer/KM forphosphotransfer to VanR compared to PhoB. No phosphotransferwasdetectable to three other E. coli response regulators, OmpR,ArcB, or CreB, providing some sense of theselectivity against two-component regulatory system cross-talk. Inthe phosphotransfer from P-VanS toPhoB and VanR, there was evidence of competition between water, to givePi, and the specific aspartyl
-COO- moiety of either PhoB or VanR, with about 25%of the initial flux generating inorganic phosphate.The kinetics of phosphotransfer from P-VanS to VanR werecomplicated by inhibition by free VanS, butthe inhibition pattern could be modeled to yield aKD of 30 nM for VanR binding to free VanS,anaffinity similar to that of the CheA-CheY pair in E.coli chemotaxis.
M were readily determined. The correspondingkxfer of 96 min-1 forphosphotransfer fromP-VanS to VanR required rapid quench kinetics. AKM of 3 M was estimated for VanR, leading toa104-fold preference inkxfer/KM forphosphotransfer to VanR compared to PhoB. No phosphotransferwasdetectable to three other E. coli response regulators, OmpR,ArcB, or CreB, providing some sense of theselectivity against two-component regulatory system cross-talk. Inthe phosphotransfer from P-VanS toPhoB and VanR, there was evidence of competition between water, to givePi, and the specific aspartyl-COO- moiety of either PhoB or VanR, with about 25%of the initial flux generating inorganic phosphate.The kinetics of phosphotransfer from P-VanS to VanR werecomplicated by inhibition by free VanS, butthe inhibition pattern could be modeled to yield aKD of 30 nM for VanR binding to free VanS,anaffinity similar to that of the CheA-CheY pair in E.coli chemotaxis.