Determining the Relative Activities of Wild-Type Mycobacterium tuberculosis Indole-3-Glycerol Phosphate Synthase and Mutants E57A and E219A With an Investigation Into the Relationship Between Wild-Type Mycobacterium tuberculosis IGPS Activity and pH

The relationship between wild-type protein indole-3-glycerol phosphate synthase (IGPS) activity and pH was investigated through the formation of pH activity profiles, and the relative catalytic activities of wild-type Mycobacterium tuberculosis and mutants E57A and E219A were also deduced in this work. The activity of each enzyme variant was determined by reacting native substrate 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate with enzyme buffered at pH 7.5 in a cuvette and using a fluorometer to detect the increase in fluorescent signal produced by the formation of fluorescent product indole-3 glycerol phosphate. Work with wild-type IGPS showed the enzyme to have a kcat of 1.26 ± 1.75 (s-1). A pH profile of wild-type IGPS was formed by allowing the reaction to proceed in buffers that maintained various pH levels between pH 5.0 and 9.5. Slopes of the activity trends formed by this method were normalized to the largest slope and plotted against pH to reveal the general pH activity profile. A plot of the averaged normalized slope values showed IGPS to have optimal enzymatic activity around 7.2 with pKa values of 6.5 ± 0.1 for pKa1 and 7.9 ± 0.1 for pKa2