LOOP DYNAMICS IN THE FUNCTION OF E. COLI INDOLE GLYCEROL PHOSPHATE SYNTHASE

Open Access
Author:
Padaki, Ajay Shridar
Area of Honors:
Chemistry
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • David D Boehr, Thesis Supervisor
  • Scott Trent Feldman, Honors Advisor
  • Howard M Salis, Faculty Reader
Keywords:
  • Enzyme Kinetics
Abstract:
A co-evolving interaction occurs between the â1á1 and â2á2 loops of Escherischia coli indole-3-glycerol phosphate synthase (ecIGPS) that may help direct enzyme catalysis. The interaction specifically occurs between Lys56 (â1á1) and Gln94 (â2á2) and is hypothesized to function in rearranging the extended formation of the 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5–phosphate (CdRP) substrate. To gain more insight into this interaction, Lys56 and Gln94 were separately mutated to Ala. The mutant enzymes were characterized in terms of their Michaelis-Menten steady-state kinetics, pH dependence, and solvent viscosity effects. While the kcat Lys56Ala IGPS decreased only 13.6% compared to wild-type (WT) IGPS, the KM, a measure of substrate binding affinity, was almost four times weaker than the WT value. In contrast, Gln94Ala IGPS displayed a slightly higher KM (41.7% greater) than the WT IGPS value, but displayed a much lower kcat (70.6% lower than the WT). The resulting catalytic efficiencies (kcat/KM) of the mutant IGPS enzymes were similarly low with a 4.5 fold decrease compared to WT IGPS. The increase in the Gln94Ala IGPS kcat of 555% as the temperature increased from 25 oC to 37 oC resulted in a more similar value to the WT IGPS. The tremendous impact of the temperature on the mutant’s catalysis speed suggests that conformational flexibility may help to mitigate the hindrances imposed by the mutated residue. While distinctions between the WT IGPS and mutant IGPS were observed in Michaelis-Menten dynamics, no such trends were observed in the pH or solvent viscosity effect (SVE) enzyme assays. Both Gln94Ala and WT IGPS displayed a bell-shaped pH dependence with pKa values close to 6.8 and 8.7 for the ascending and descending limbs of the pH rate profile, respectively. The distinct limbs are consistent with general base and acid catalysis, as previously proposed for the IGPS enzymatic mechanism. The lack of any observable SVE indicates the chemical step is likely the rate-determining step, which simplified the analysis of k¬cat and KM values. The low KM of the Lys56Ala mutant and the slow catalysis of the Gln94Ala mutant suggest that the interaction between the â1á1 and â2á2 loops is intimately involved in the binding and ring enclosure of the CdRP substrate.