Abstract

Chorismate mutase - prephenate dehydrogenase (CM-PD) is a bifunctional enzyme that catalyzes two sequential reactions in the tyrosine biosynthetic pathway of E. coli . Three cysteine residues are found within each monomer of dimeric CM-PD. The studies in Chapter 2 help to define their role in the structure of the enzyme, the chemistry of the reactions, and in the relationship between the sites at which the two reactions occur. Site-directed mutagenesis performed on Cys95, Cys169 and Cys215 indicated that only Cys215 is important for the activity of CM-PD and is most important in the binding of prephenate. However, comparative modeling studies indicate that Cys215 may be at the active site of prephenate dehydrogenase and help orient an important catalytic group, His197. Chemical modification of the enzyme with cysteine-specific reagents, iodoacetamide and Ellman's reagent (DTNB), resulted in the loss of both mutase and dehydrogenase activities. Inactivation of wild-type and variant forms of prephenate dehydrogenase suggested that one cysteine (Cys215) is protected against alkylation by NAD + plus tyrosine, prephenate and, surprisingly, an inhibitor that mimics the transition state of the mutase reaction. Comparing the effects of alkylation of wild-type and K37Q by iodoacetamide showed that the protective effects of prephenate and the mutase transition state analog were due to their binding to the mutase site rather than the dehydrogenase site. Time-dependent chemical modification followed by peptide mapping indicated that Cys215 is the most reactive and/or accessible cysteine, followed by Cys95 and Cys 169. In Chapter 3, ESI-mass spectrometry in conjunction with chemical modification studies were used to determine the reactivity and ionization state of Lys37 of CM-PD.