Studying chemical modification and limited proteolysis of E. coli citrate synthase by mass spectrometry

• Main Author: Sadler, Gillian Wendy
• Format: Others
• Language: en; en_US
• Published: 2007
• Online Access: http://hdl.handle.net/1993/2432

Citrate synthase (CS) of 'E. coli' is strongly inhibited by the binding of NADH to an allosteric site distinct from the enzyme's active site. Previous chemical modification and mutagenesis studies have implicated a reactive cysteine, C206 as being at or near the allosteric site. This thesis details an attempt to clarify the location of the allosteric site using the photoaffinity label 2-azidoATP. Electrospray ionization time-of-flight mass spectrometry (ESI TOFMS), and capillary electrophoresis interfaced with mass spectrometry were used to map the locations of photolabel incorporation in unfractionated tryptic digests of CS. Although several putatively labeled peptides were noted, the best candidate was a peptide of mass 1666.8 Da, which could arise from one or both of tryptic fragments T17 (1146.48 Da, corresponding to residues 168-177) and T15-16 (1147.68 Da, corresponding to residues 158-167). Both of these peptides are structurally close to C206. Mass spectrometry was also applied to the study of several other characteristics of 'E. coli' CS previously examined by more conventional means. Chemical modifications targeted at the two reactive sulfhydryl groups of CS were mapped in unfractionated digests by matrix assisted laser desorption ionization (MALDI) TOFMS. Limited proteolysis of CS by trypsin and subtilisin was monitored by ESI TOFMS. An unexpected result of these experiments was the "all or nothing" breakdown of CS, with the production of mostly small peptides and very few large intermediate proteolytic fragments. The behaviour of the subunit equilibrium of CS in the presence of an allosteric activator was also studied using ESI TOFMS. Finally, the results of undergraduate project course work carried out on two allosteric site mutants and the manner in which these results relate to those previously obtained for other CS mutants, are also discussed.