1CM3

HIS15ASP HPR FROM E. COLI

Summary for 1CM3

• Entry DOI: 10.2210/pdb1cm3/pdb
• Related: 1CM2
• Descriptor: HISTIDINE-CONTAINING PROTEIN (2 entities in total)
• Functional Keywords: phosphotransferase, transferase
• Biological source: Escherichia coli
• Cellular location: Cytoplasm: P0AA04
• Total number of polymer chains: 1
• Total formula weight: 9106.27

Authors

Napper, S.,Waygood, E.B.,Delbaere, L.T.J. (deposition date: 1999-05-13, release date: 2000-05-17, Last modification date: 2024-04-03)

Primary citation

Napper, S.,Delbaere, L.T.,Waygood, E.B.
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
J.Biol.Chem., 274:21776-21782, 1999

PubMed Abstract: The active site residue, His(15), 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 His(15) --> Asp HPr is increased 10-fold and V(max) is decreased 1000-fold compared with wild type HPr. The phosphorylation of Asp(15) 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 His(15) --> 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 Asn(12) in an internal cyclization with Asp(15) was eliminated by the Asn(12) --> Ala mutation in His(15) --> AspHPr. Asn(12) 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 Nepsilon(2) atom of His(15), but elimination of the hydrogen bond has only a 4-fold decrease in k(cat)/K(m).

PubMed: 10419492
DOI: 10.1074/jbc.274.31.21776

Experimental method

X-RAY DIFFRACTION (1.6 Å)