INVOLVEMENT OF VARIOUS AMINO-TERMINAL 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 carnosu s was modified by site-directed mutagenesis of the corresponding ptsH gene in order to analyse the importance of amino acids which were supp osed to be part of the active centre of the protein. Three residues wh ich are conserved in all HPrs, Arg17, Pro18 and Glu84, were mutated: A rg17 was changed to His (17RH) and Pro18 and Glu84 were changed into A la (18PA and 84EA). In addition, Leu86 was changed into Ala (86LA) and one mutant protein was missing the last six residues of the HPr (DELT A83). The wild type gene and all mutant genes were overexpressed and t he gene products purified to homogeneity. Three-dimensional structures of wild type and mutant proteins were monitored by NMR spectroscopy. All rive mutant HPrs had native conformations. The ATP-dependent HPr k inase 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 H Prs 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 enzym e activity of the HPr 17RH was significantly improved at low pH. NMR p H-titration experiments showed that Arg17 is not responsible for the l ow pK(a) of the active centre His15 but this positively charged residu e is essential in this position for the HPr activity.