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).
Highlights
Histidine-containing protein, HPr, is a small phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS).1 The PTS is a bacterial system that transports and phosphorylates many sugars and is involved in major regulatory events of carbohydrate metabolism
His15 3 Asp HPr Can Act as a Phosphoacceptor—The following substitutions for His15 in E. coli HPr were made: alanine, asparagine, aspartate, cysteine, glutamate, glutamine, serine, threonine, and tyrosine
Except for His15 3 Asp HPr, none of these mutations showed any detectable activity when tested for activity with enzyme I by: (a) a spectrophotometric assay for enzyme I activity [43]; (b) [32P]-protein labeling by PEP detected by SDS-polyacrylamide gel electrophoresis and autoradiography [42]; (c) a gel shift of a band on an isoelectric focusing (IEF) gel because of the introduction of the phosphoryl group
Summary
Histidine-containing protein, HPr, is a small phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS is a bacterial system that transports and phosphorylates many sugars and is involved in major regulatory events of carbohydrate metabolism (see reviews in Refs. 1– 4). HPr was first described by Kundig et al [5] when the function of accepting and donating a phosphoryl group from enzyme I to the sugar-specific enzymes II was established for Escherichia coli. These events result in phosphoprotein formation with a N⑀2-P-histidine in both enzyme I [6] and the enzyme IIAsugar domains [7] and a more unstable N␦1-P-histidine in HPr [8, 9]. Asn in E. coli HPr has been investigated because it is a site of deamidation, which occurs through the formation of a succinimide to form aspartate and isoaspartate at residue 12 [19]. The P-aspartyl residue leads to a spontaneous chemical rearrangement with the characteristics of catalyzed isoimide formation
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