Abstract
Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.
Highlights
Histidine-containing protein, HPr, of the Esche- Meadow et al (1990))
Terized as a substrate, it probably does participate in a catalytic manner to facilitate the phosphoryl transfer
The structuresof HPrs fromBacillus subtilis protein of the phosphoeno1pyruvate:sugar phosphotransferase and Streptococcus faecalk (Wittekind et al, 1990, 1992; Herzsystem (PTS).It is the acceptor of a phosphoryl group from berg et al, 1992; Jia et al, 1993)have theC-terminal aenzyme I, and the donor of the group toa sugar-specific IIA carboxyl away from the active site
Summary
General Properties of Mutant Proteins-To investigate the role that Arg-17 contributes to theactivity of HPr, thefollowing site-directed mutants were made: substitution with other basic residues (histidine and lysine, R17H and R17K); an acidic residue (glutamate, R17E); and smaller, neutral residues (cysteine, glycine, and serine, R17C, R17G, and R17S). On the basis of studies with modified histidines, Hultquist (1968) concluded that theincreased rates of phosphohydrolysis and increased instability were due to theinteraction of the free cyamino group of the amino acid with the phosphoryl group Because this amino group is eliminated by peptide bond formation, the fact that the N*'-P-histidine in P-HPr had phosphohydrolysis rates in excess of the free N*'-P-histidine has been attributed tothe interaction between Arg-17 guanido group and the phosphoryl group (Waygood et al, 1985). 17 mutants, and, apart from R17H (Fig. lB),all the substitutions gave a similar effect, yielding a considerable reduction of rates between pH 2 and 8.5 (Fig. lA).Except for R17H, the "apparent pK,," of this process appears to shift from about 7.7 for wild type to values closer to 8.0 to 8.2, consistent with the more carefully determined pK, values below. The phosphohydrolysis rates observed for these mutants were similar to those found for the free N*'-P-
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