E. coli HPr

Figure 6. Elution profile obtained on examination of the gel permeation behavior of 14C acetylated S. aureus Factor III. Cytochrome-c and fluorescein labeled E. coli HPr were included in the applied sample as internal standards. Absorbance at 410 nm (cytochrome-c), fluorescein fluorescence (HPr), and radioactivity (Factor III) are plotted vs. fraction number. Inset shows the elution profiles for the external (blue dextran) and internal (fiuoroglycine) column markers. Data in this figure are from unpublished work (20).

Ill, which had been radioactively labeled by 14C acetylation. Cyto-chrome-c and fluorescein labeled E. coli HPr, a protein of known sequence, have been added to the sample as internal molecular weight standards. The Mapp determined for Factor III by gel permeation in 6M GuHCl, 11,000 300, is in good agreement with the value obtained from sedimentation equilibrium in 6M GuHCl-O.lM 2-mercaptoethanol, 12,000 (20). 

FIGURE 10.26 Glucose transport in E. coli is mediated by the PEP-dependent phosphotransferase system. Enzyme I is phosphorylated in the first step by PEP. Successive phosphoryl transfers to HPr and Enzyme III in Steps 2 and 3 are followed by transport and phosphorylation of glucose. Enzyme II is the sugar transport channel. 

As shown in Fig. 1, the function of E-III is to transfer the phosphoryl group from P-HPr to E-II. P-phosphorylated peptides have been isolated from a number of E-III species and A domains of S. carnosus and S. aureus IIl [27], E. coli II [28],... 

The fructose-specific PTS in R. sphaeroides is simpler than the one in E. coli or S. typhimurium in that it consists of only two proteins. Besides the fructose specific ll , a class II enzyme, there is only one cytoplasmic component called soluble factor (SF) [48]. We now know that SF consists of IIl , HPr and E-I covalently linked [109]. 11 and SF form a membrane-bound complex whose association-dissociation dynamics is much slower than the turnover of the system. Therefore, the complex is the actual catalytic unit in the overall reaction and P-enolpyruvate is the direct phosphoryl group donor [102],... 

Enzyme I from E. coli catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to a phospho carrier protein (HPr). A phosphoryl group is transferred to a dimeric form of the enzyme. Mg2+ or Mn2+ is necessary for stabilization of the dimer and for activity, although it is not known whether these functions are carried out at separate sites.310 A scheme is shown in equations (5) and (6). 

Section IV,B,2 introduces the idea that chemical-shift anisotropy could become an important relaxation mechanism for phosphorus at hi field. Vogel et al. (1982) have published the field dependence of the linewidth of the histidine 3-iV-phosphate of succinyl-CoA synthetase and the serine phosphate of glycogen phosphorylase a. They found that the CSA mechanism did, indeed, dominate at 6.3- and 9.3-T fields, contributed substantially at 4.7 T, and contributed about 25% of the total relaxation at 2.1 T (36.4 MHz). Vogel etal. 1982) estimated for Escherichia coli sucdnyl-CoA synthetase (MW 140,(XX)) a hydrated radius of 37 A and an isotropic correlation time of 44 ns. These numbers are referenced to the phosphorus linewidth (sensitive only to overall motion) using an analysis very similar to that mentioned in Section IV,B on DNA phosphorus relaxation. In contrast, the E. coli phosphoryl carrier protein HPr (MW 9(KX)) has a calculated hydrated radius of 17 A and a correlation time of 4.2 ns, according to Vogel et al. (1982). 

Napper, S. Delbaere, L.T.J. Waygood, E.B. The aspartyl replacement of the active site histidine in histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate sugar phosphotransferase system can accept and donate a phosphoryl group. Spontaneous dephosphorylation of acyl-phosphate autocatalyzes an internal cyclization. J. Biol. Chem., 274, 21776-21782 (1999)... 

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