Involvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins.
The phosphocarrier HPr (heat stable protein) of Staphylococcus carnosus was modified by site-directed mutagenesis of the corresponding ptsH gene in order to analyse the importance of amino acids which were supposed to be part of the active centre of the protein. Three residues which are conserved in all HPrs, Arg17, Pro18 and Glu84, were mutated: Arg17 was changed to His (17RH) and Pro18 and Glu84 were changed into Ala (18PA and 84EA). In addition, Leu86 was changed into Ala (86LA) and one mutant protein was missing the last six residues of the HPr (delta 83). The wild type gene and all mutant genes were overexpressed and the gene products purified to homogeneity. Three-dimensional structures of wild type and mutant proteins were monitored by NMR spectroscopy. All five mutant HPrs had native conformations. The ATP-dependent HPr kinase can phosphorylate all HPr derivatives at Ser46. The PTS activity of the amino-terminal HPr mutant proteins 17RH and 18PA was different compared to wild type HPr. In contrast, the carboxy-terminal mutant HPrs possessed a similar enzyme activity to the wild type HPr. The 17RH and 18PA HPrs with substitution near the active centre His15 showed a very slow phosphorylation by enzyme I but the further transfer of the phosphoryl group to enzyme III was also strongly inhibited. The enzyme activity of the HPr 17RH was significantly improved at low pH. NMR pH-titration experiments showed that Arg17 is not responsible for the low pKa of the active centre His15 but this positively charged residue is essential in this position for the HPr activity.
Kruse
R
R
Abteilung für Biologie, Ruhr-Universität Bochum, Germany.
Hengstenberg
W
W
Beneicke
W
W
Kalbitzer
H R
HR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D015870:Gene Expression; D006639:Histidine / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D013210:Staphylococcus / Q000201:enzymology*
D001426:Bacterial Proteins; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr