Structure and function of enzyme I of the PTS
| dc.contributor.committeeMember | Waygood, Edward | en_US |
| dc.creator | Brokx, Stephen John | en_US |
| dc.date.accessioned | 2004-10-21T00:28:44Z | en_US |
| dc.date.accessioned | 2013-01-04T05:06:19Z | |
| dc.date.available | 2000-09-01T08:00:00Z | en_US |
| dc.date.available | 2013-01-04T05:06:19Z | |
| dc.date.created | 2000-09 | en_US |
| dc.date.issued | 2000-09-01 | en_US |
| dc.date.submitted | September 2000 | en_US |
| dc.description.abstract | The phosphoenolpyruvate: sugar phosphotransferase system (PTS) is responsible for the uptake and concomitant phosphorylation of many sugars in several species of bacteria. The first step of the PTS involves the phosphorylation of histidine containing phosphocarrier protein (HPr) by enzyme I (E.C. 2.7.3.9), with phosphoenolpyruvate (PEP) serving as the phosphoryl donor. Enzyme I has logically been viewed as a potential target for regulation of the PTS. This thesis presents important information regarding the structure and function of enzyme I of Escherichia coli and Salmonella typhimurium. Fluorescence polarization analysis, although incomplete, showed that the interaction of HPr with enzyme I and with enzyme IIA glc are of low affinity, with a Kd of roughly 10-100 [mu]M. An enzyme I binding site on HPr was determined by a kinetic assay, using site-directed mutants of HPr as substrates for enzyme I. This site of interaction agreed very well with that found in the NMR solution structure of the complex of HPr with the N-terminal domain of enzyme I (Garrett 'et al'., 1999), with a few important differences. Genes encoding mutant enzymes I were cloned from 'S. typhimurium ' strains and the purified proteins were analyzed. The Arg126Cys mutant was defective in phosphotransfer, while the Gly356Ser and Arg375Cys mutants were defective in dimerization and PEP-binding. Intragenic complementation was observed between purified Arg126Cys and Gly356Ser or Arg375Cys enzymes I, through formation of heterodimers. This heterodimers were unstable, and stability depended upon dilution of the enzyme and PEP concentrations. Other site-directed mutants of ,E. coli enzyme I were created, which indicated the importance of the residues Asn352 and Leu355 in dimerization, and Arg296 in PEP-binding. These data led to the conclusions that dimerization and PEP-binding of enzyme I are closely linked cooperative events, and that the monomer:dimer equilibrium of enzyme I, with the dimer being the most active form, may have significant physiological importance. Regulation of the monomer:dimer equilibrium may provide a key target for modulation of the activity of enzyme I, and thus regulation of the PTS. | en_US |
| dc.identifier.uri | http://hdl.handle.net/10388/etd-10212004-002844 | en_US |
| dc.language.iso | en_US | en_US |
| dc.subject | biochemistry | en_US |
| dc.title | Structure and function of enzyme I of the PTS | en_US |
| dc.type.genre | Thesis | en_US |
| dc.type.material | text | en_US |
| thesis.degree.department | Biochemistry | en_US |
| thesis.degree.discipline | Biochemistry | en_US |
| thesis.degree.grantor | University of Saskatchewan | en_US |
| thesis.degree.level | Doctoral | en_US |
| thesis.degree.name | Doctor of Philosophy (Ph.D.) | en_US |