Investigating the ionization state and structure/function relationship of ionizable residues in Escherichia coli chorismate mutase -- prephenate dehydrogenase and the catalytic trimer of aspartate transcarbamylase

Modern mass spectrometry has gained tremendous attention III the field of enzymology, in particular as a tool for dissecting enzyme mechanism. In this thesis we use mass spectrometry in combination with chemical modification to help define the roles and ionization states of active site residues that are important for enzyme activity. Enzymes of particular interest are chorismate mutase-prephenate dehydrogenase (CM-PD), a bifunctional enzyme from Escherichia coli that catalyzes two consecutive steps in tyrosine biosynthesis, and the catalytic trimer of E. coli aspartate transcarbamylase (ATCase), which catalyzes the first committed step in the biosynthesis of pyrimidines. All three enzymes are of industrial or medical value as targets for the design of inhibitors that can act as either anti-neoplastic agents, antimicrobial agents or herbicides.;In E. coli, mutase activity is associated with the N-terminal domain of CM-PD. pH rate profiles of the mutase reaction show that there are two residues whose ionization states are important for catalysis and/or substrate binding. Studies examining the rates of chemical modification of the enzyme with a lysine-specific reagent and the stoichiometry of modification indicate that only one lysine residue per monomer, Lys37, is particularly reactive. Peptide mapping was used to determine the pKa of the epsilon-amino group of Lys37 to be 7.5. Chemical modifications, kinetic and binding studies on Lys37Gln, an inactive variant, show that Lys37 is critical for CM activity. We propose that Lys37 participates in catalysis by protonating the ether oxygen of chorismate in the reaction's transition state.;ATCase is composed of 3 regulatory dimers and 2 catalytic trimers. Treatment with mercurial reagents dissociates the regulatory subunits (RSU) from the catalytic subunits (CSU) without compromising their functions. An improved purification scheme is outlined for the different subunits. Biophysical studies on an inactive CSU variant, Ser52Cys, indicated that the substitution resulted in a more thermally stable enzyme. Chemical modification by cysteine-specific reagents and mass spectrometric analysis indicated that Cys52 is very reactive/accessible and possesses a pKa of ∼5.6. The unusual characteristics of the Ser52Cys variant are attributed to the markedly depressed p Ka of Cys52. The biophysical reasons for the ionization state of Cys52 are discussed in terms of the crystal structure of the unliganded E. coli CSU.;Three cysteine residues are found within each monomer of the dimeric CM-PD. Site-directed mutagenesis and kinetic analysis of variants of Cys95, Cys169 and Cys215 indicated that only Cys215 is important for both CM and PD activities. Chemical modification with cysteine-specific reagents, iodoacetamide and Ellman's Reagent, resulted in the loss of both activities but only Cys215 is protected against alkylation by ligands of the reaction and therefore near or in the active site. Time-dependent chemical modification followed by peptide mapping indicated that Cys95 is the most reactive and/or accessible cysteine followed by Cys215 and Cys169. The results are discussed in terms of a structural model of the E. coli PD domain; Cys215 is near His245, which appears to help orient the catalytic base of the dehydrogenase reaction, His197.