Purification and Characterization of HisP, the ATP-binding Subunit of a Traffic ATPase (ABC Transporter), the Histidine Permease of Salmonella typhimurium

• Published in: The Journal of biological chemistry 1997-10, Vol.272 (44), p.27745-27752
• Main Authors: Nikaido, Kishiko, Liu, Pei-Qi, Ames, Giovanna Ferro-Luzzi
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.44.27745

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 aKd value of 205 μm, 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 byN-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.