Roles of Large Loops in Catalytic Versatility of Catalase-Peroxidases: Significance of Peripheral Structures in Improvising Enzyme Functions
Type of Degreedissertation
DepartmentChemistry and Biochemistry
MetadataShow full item record
Catalase-peroxidases are bifunctional enzymes that catalyze the removal of hydrogen peroxide by two distinct pathways (catalase and peroxidase). They are central to antibiotic resistance in Mycobacterium tuberculosis and may be virulence factors in several dangerous human pathogens. These enzymes also hold much promise for engineering new enzymes to combat long-standing problems (e.g., environmental contamination by toxic pollutants). Currently, understanding of catalase-peroxidase structure and function is lacking to facilitate new drug development and enzyme engineering. The purpose of the research described in this dissertation is to understand the molecular basis for the unique catalytic abilities of catalase-peroxidases to fully capitalize on their potential. The versatile catalytic abilities of these enzymes arise from an active site that is normally restricted to one activity (i.e., peroxidase). This is facilitated by three structures which are quite distant from the active site: a C-terminal domain and two large loops (LL1 and LL2). Given that catalase-peroxidases have two large loops absent from their fellow peroxidases, it is reasonable to suggest that these loops may serve to fine tune the active site for bifunctionality. Indeed, deletion of any of these structures has a substantial impact on catalase-peroxidase active site environment and catalytic function (e.g., selective loss of catalase activity). The main emphasis of this dissertation is to explore the roles of these two large loops i) by identifying additional roles of LL1 in the unique catalytic properties of KatGs, and ii) by targeting the intersubunit interactions between LL2 apex and C-terminal domain. Site directed and deletion variants targeting LL2 apex and its length and various portions of LL1 (Y226F KatG, KatG∆209-228, KatG∆200-214, KatG∆LL1) were generated and investigated by heme environmental spectroscopy, steady-state and transient-state kinetic techniques. For LL1, all the variants including tyrosine showed a complete loss of catalase activity but the deletion variants showed substantial increase in peroxidase activity compared to wild type (wt KatG) and Y226F KatG. This increase in peroxidase activity was traceable to rapid reduction of high oxidation state intermediates by exogenous electron donors, and coincident prevention of enzyme inactivation by peroxides. The steady-state kinetic parameters of KatG∆200-214 were comparable to wt KatG. In case of LL2, deletion variants showed near to complete loss of catalase activity with variable effects on peroxidase activity. The peroxidase activity with respect to H2O2 showed a substantial rate of turnover (even in excess of wt KatG) at sufficiently high H2O2 concentrations, along with two orders of magnitude lower apparent second order rate constants suggesting deficiency in the correct association of H2O2 in the active site leading towards it reduction to H2O. However, peroxidase kinetic parameters with respect to ABTS suggested that electron transfer to the heme by exogenous electron donors is unaffected and in some cases even enhanced. Concomitant with the shift in activity was a change in heme coordination from predominantly hexacoordinate high-spin to predominantly pentacoordinate high-spin. In contrast to deletion variants, the apex substitution variants of LL2 showed similar spectroscopic characteristics compared to wt KatG with little to no effect on the catalytic activities. The results of these studies emphasize the fact that both large loops despite of their peripheral locations serve to fine-tune the active site for its bifunctionality by acting as a gate keeper (LL1) to limit the typical peroxidase activity in favor of catalase activity and by maintaining the unique interaction occurring at 30Å from the active site between LL2 and C-terminal domain.
- Shalley N Kudalkar Dissertation.pdf.txt
- Shalley N Kudalkar Dissertation.pdf