J Biol Chem, Vol. 274, Issue 31, 21776-21782, July 30, 1999

The Aspartyl Replacement of the Active Site Histidine in Histidine-containing Protein, HPr, of the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System Can Accept and Donate a Phosphoryl Group
SPONTANEOUS DEPHOSPHORYLATION OF ACYL-PHOSPHATE AUTOCATALYZES AN INTERNAL CYCLIZATION

From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada

The active site residue, His¹⁵, in histidine-containing protein, HPr, can be replaced by aspartate and still act as a phosphoacceptor and phosphodonor with enzyme I and enzyme IIA^glucose, respectively. Other substitutions, including cysteine, glutamate, serine, threonine, and tyrosine, failed to show any activity. Enzyme I K_m for His¹⁵  Asp HPr is increased 10-fold and V_max is decreased 1000-fold compared with wild type HPr. The phosphorylation of Asp¹⁵ led to a spontaneous internal rearrangement involving the loss of the phosphoryl group and a water molecule, which was confirmed by mass spectrometry. The protein species formed had a higher pI than His¹⁵  Asp HPr, which could arise from the formation of a succinimide or an isoimide. Hydrolysis of the isolated high pI form gave only aspartic acid at residue 15, and no isoaspartic acid was detected. This indicates that an isoimide rather than a succinimide is formed. In the absence of phosphorylation, no formation of the high pI form could be found, indicating that phosphorylation catalyzed the formation of the cyclization. The possible involvement of Asn¹² in an internal cyclization with Asp¹⁵ was eliminated by the Asn¹²  Ala mutation in His¹⁵  AspHPr. Asn¹² substitutions of alanine, aspartate, serine, and threonine in wild type HPr indicated a general requirement for residues capable of forming a hydrogen bond with the N² atom of His¹⁵, but elimination of the hydrogen bond has only a 4-fold decrease in k_cat/K_m.