Studies on the chemistry of enzyme active sites

• Main Author: Krell, Tino
• Published: University of Glasgow 1996
• Subjects: 572; QH345 Biochemistry
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360309

A major part of this thesis involved using electrospray mass spectrometry to monitor the chemical modification of amino acid side chains in enzymes. The technique was used specially to locate active site residues in shikimate pathway enzymes and to monitor the dephosphorylation of a phospho enzyme immediate. Firstly, site-specific chemical modification in combination with mass spectrometry was used to identify Arg-23 in Streptomyces coelicolor type II dehydroquinase (DHQ) as a residue essential for enzyme function. This residue was replaced by lysine, glutamine and alanine residues using site-directed mutagenesis. All the mutants were shown to have much lower turn over numbers as well as lower Km values in comparison to the native enzyme. This makes a role for Arg-23 in substrate binding unlikely. A catalytic role for this residue in stabilising a negatively charged enolate transition state is proposed since the mutant R23A was found to be 10 times less active than R23K and R23Q. Furthermore, Tyr-28 of S. coelicolor DHQ and Arg-213 of Escherichia coli type I DHQ have been shown to be in or near the active site. Secondly, mass spectrometry was used to monitor the dephosphorylation of phosphorylated forms of phosphoglycerate mutases (PGAM). The phosphorylated PGAM from Saccharomyces cerevisiae was shown to be at least 35 times more stable than the enzyme from Schizosaccharomyces pombe which does not contain a C-terminal segment of 14 amino acids which is probably responsible for the differences in stability. The phosphorylated mutant H163Q mutant of S. cerevisiae PGAM appeared to be at least 400 times more stable than the native enzyme. Thirdly, chemical modification and mass spectrometry were used to identify active site residues in E. coli shikimate dehydrogenase (SHD). Two lysine residues were shown to be in or near the active site.