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  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

Kruse, R., Hengstenberg, W., Beneicke, W., & Kalbitzer, H. R. (1993). 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. Protein Engineering, 6(4), 417-423. doi:10.1093/protein/6.4.417.

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Kruse,, Regina, Author
Hengstenberg,, Wolfgang, Author
Beneicke, Wolfgang1, 2, Author           
Kalbitzer, Hans Robert2, Author           
Affiliations:
1IT Services, Max Planck Institute for Medical Research, Max Planck Society, ou_persistent22              
2Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              

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Free keywords: HPr of Staphylococcus carnosus/NMR spectroscopy/phosphotransferase system/ptsH gene/site-directed mutagenesis
 Abstract: 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, Argl7, Prol8 and Glu84, were mutated: Argl7 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 (δ83). 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 car boxy-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.

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Language(s): eng - English
 Dates: 1993-02-021992-09-161993-03-101993-06-01
 Publication Status: Issued
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 665090
DOI: 10.1093/protein/6.4.417
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Title: Protein Engineering
Source Genre: Journal
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Publ. Info: Oxford [England] : IRL Press
Pages: - Volume / Issue: 6 (4) Sequence Number: - Start / End Page: 417 - 423 Identifier: ISSN: 0269-2139
CoNE: https://pure.mpg.de/cone/journals/resource/111074106465000