Purification and characterization of HisP, the ATP-binding subunit of a traffic ATPase (ABC transporter), the histidine permease of Salmonella typhimurium. Solubility, dimerization, and ATPase activity

J Biol Chem. 1997 Oct 31;272(44):27745-52. doi: 10.1074/jbc.272.44.27745.

Abstract

The nucleotide-binding subunit, HisP, of the histidine permease, a traffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP(His6)) allows a rapid and effective metal affinity purification, giving a 30-fold purification with a yield of 50%. HisP(his6) is indistinguishable from underivatized HisP when incorporated into the permease membrane-bound complex, HisQMP2. Purified HisP(his6) has a strong tendency to precipitate; 5 mM ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP(his6) is active as a dimer, binds ATP with a Kd value of 205 microM, and hydrolyzes it at a rate comparable to that of HisQMP2; in contrast to the latter, it does not display cooperativity for ATP. HisP(his6) has been characterized with respect to substrate and inhibitor specificity and various physico-chemical characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co2+ and Mn2+ being more effective than Mg2+ at lower concentrations but inhibitory in the higher concentration range. In contrast to the intact complex, HisP(his6) is not inhibited by vanadate but is inhibited by N-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP-Binding Cassette Transporters / metabolism*
  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / metabolism*
  • Amino Acid Transport Systems, Basic*
  • Bacterial Proteins*
  • Biological Transport, Active
  • Dimerization
  • Membrane Transport Proteins / isolation & purification*
  • Membrane Transport Proteins / metabolism
  • Plasmids
  • Protein Binding
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Salmonella typhimurium / enzymology*
  • Solubility

Substances

  • ATP-Binding Cassette Transporters
  • Amino Acid Transport Systems, Basic
  • Bacterial Proteins
  • Membrane Transport Proteins
  • Recombinant Proteins
  • histidine permease, Bacteria
  • Adenosine Triphosphate
  • Adenosine Triphosphatases