Phosphorylation sites of HPr
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The histidine-containing protein (HPr) is a central phosphotransfer component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. There are two HPr phosphorylation events investigated in this thesis. Firstly, BPr from Gram-positive species may undergo a regulatory phosphorylation of an absolutely conserved Ser46 residue. There are numerous metabolic consequences to this phosphorylation, including inducer exclusion and expulsion, inhibition of PTS sugar uptake and catabolite repression. While HPr from Gram-negative sources cannot undergo phosphorylation of Ser46 'in vivo' or ' in vitro' it is possible to mimic the phosphorylation through the Ser46Asp mutation. To determine the structural consequences of the mutation the crystallographic structure of the 'E. coli'. Ser46Asp HPr was determined at 1.5 Å resolution. The structure revealed that no significant structural rearrangements are induced by the mutation and the inability to accept phosphotransfer from Enzyme I is due to electrostatic disruption of the interaction of these proteins. Phosphorylation of an absolutely conserved His15 for the purpose of phosphotransfer represents the second phosphorylation event to be investigated. The absolute requirement for histidine at the 15 position was investigated through mutagenesis. The mutation of His15Asp of 'E. coli' HPr was able to accept a phosphoryl group from Enzyme I and further transfer the phosphoryl group to Enzyme IIAglc. None of the other mutations of the fifteen position were able to be phosphorylated. The His15Asp mutant had a Vmax of 0.1% and a ten-fold increase in Kin with respect to wild type HPr. As a consequence of the phosphorylation of His15Asp HPr a third protein species of higher pI than the original protein was identified. This high pI species seemed to share numerous similarities to succinimides which are known to be involved in deamidation. The inability to detect the known degradation products of succinimides suggested that the high pI species may involve isoimide formation. Isoimides have been proposed, but never experimentally demonstrated in proteins. A mechanism through which the phosphoacyl intermediate may catalyze isoimide formation is proposed. In addition the potential involvement of isoimide formation as a mechanism in physiological regulatory signaling is discussed.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeDelbaere, Louis T. J.
Copyright DateSeptember 1999