1cm3
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HIS15ASP HPR FROM E. COLI
OverviewOverview
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).
About this StructureAbout this Structure
1CM3 is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
ReferenceReference
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., Napper S, Delbaere LT, Waygood EB, J Biol Chem. 1999 Jul 30;274(31):21776-82. PMID:10419492
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