Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.
Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.
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大肠杆菌磷酸烯醇丙酮酸：糖磷酸转移酶系统的含组氨酸蛋白 HPr 的活性位点中羧基末端残基的参与。
DOI：
10.1021/bi00104a006
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Waygood,EB
中科院分区：
文献类型：
--
作者：
Anderson,JW;Bhanot,P;Georges,F;Klevit,RE;Waygood,EB
材料和方法材料。限制酶、Klenow片段、T4 DNA连接酶和2 '，3'-双脱氧核苷酸来自Pharmacia和新英格兰Biolabs。放射性标记的化合物购自NEN。如Waygood和Steeves（1980）所述，从大肠杆菌菌株WA 2127中纯化酶I，该菌株含有质粒pTSHIC 9，该质粒含有基因ptsHIcrr，由B博士慷慨提供。厄尼均质E. coliPhosphoe-nolpyruvate carboxkinase是H.歌蒂酶测定。如前所述进行酶Hmanno 1、酶Hmanno 1、酶Hmanno 1和酶Hmanno 2的测定（Waygood等人，1979; Waygood & Steeves，1980）。酶IIman是因子III和酶II的复合物，其分离自在乳酸盐基本盐培养基上生长的鼠伤寒沙门氏菌菌株SB 2950的粗膜培养物（Waygood等人，1984年）。酶IImt 1和酶II”ag 2在E.大肠杆菌菌株CSH 4 trp rpsL在含有0. 2%甘露醇或0.2%Ar-乙酰葡糖胺。为了确保这些不连续测定的线性，在10和20 min时对测定进行取样。对于酶II，获得6至8个HPr浓度值（2至100 µ）的初始速度。乳酸脱氢酶偶联测定用于酶I（Waygood等人，1979年），并使用1.5和80 µ之间的9个HPr浓度值来获得初始速度数据。为了获得可再现的酶I动力学，必须注意酶I的稀释和储存（Waygood等人，1979; Waygood，1986），并使用以下程序。将酶I（约1.3mg/mL）用0.01M 2（N-吗啉代）乙磺酸（Mes）缓冲液（pH 6.8）透析，
Materials and Methods Materials. Restriction enzymes, Klenow fragment, T4 DNA ligase, and 2', 3'-dideoxynucleotides were from Pharmacia and New England Biolabs. Radiolabeled compounds were purchased from NEN. Enzyme I was purified as pre-viously described by Waygood andSteeves (1980) from Es-cherichia coli strain WA2127 containing plasmid pTSHIC9, which contained the genes ptsHIcrr, and which was generously provided by Dr. B. Erni. Homogeneous E. coli phosphoe-nolpyruvate carboxykinase was a gift from Dr. H. Goldie. Enzyme Assays. The assays for enzyme IImannoee, enzyme j jv-acetyigiucosamine^ anc¡ enzyme Hmannito1 were carried out as described previously (Waygood et al., 1979; Waygood & Steeves, 1980). Enzyme IIman was the complex of the factor III and enzyme II that is isolated in crude membrane prepa-rations of Salmonella typhimurium strain SB2950 grown on lactate minimal salts medium (Waygood et al., 1984). Enzyme IImtl and enzyme II" agwere in membranes from E. coli strain CSH4 trp rpsL grown on minimal salts media with either0. 2% mannitol or 0, 2% Ar-acetylglucosamine, respectively. In order to ensure the linearity of these discontinuous assays, the assays were sampled at both 10 and 20 min. Initial velocities were obtained for six to eight concentration values of HPr from 2 to 100 µ for the enzymes II. The lactate dehydrogenase coupled assay was used for enzyme I (Waygood et al., 1979), and nine HPr concentration values between 1.5 and 80 µ were used to obtain initial velocity data. To obtain reproducible enzyme I kinetics, care must be taken with the dilution and storage of enzyme I (Waygood et al., 1979; Waygood, 1986), and thefollowing procedures were used. Enzyme I (about 1.3 mg/mL) was dialyzed against 0.01 M 2 (N-morpholino) ethanesulfonic acid (Mes) buffer, pH 6.8, with