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Role of Distant, Intrasubunit Residues in Catalase-Peroxidase Catalysis: Tracing the Role of Gene Duplication and Fusion in Enzyme Structure and Function




Cook, Carma

Type of Degree



Chemistry and Biochemistry


In spite of being positioned 30 Å away from the active site, the C-terminal domain is essential for catalase-peroxidase (KatG) function. Addition of equimolar amounts of separately expressed and isolated C-terminal domain (KatGC) to the active-site bearing N-terminal domain (KatGN) restores KatG function. There are highly conserved interdomain residue interactions that could be essential for the reactivation of KatGN. Kinetic and spectroscopic studies indicate that the Arg 117-Asp597 (R117-D597) salt-bridge interaction is not essential for KatG catalytic function. However R117 and D597 individually appear to play a significant role in stabilizing a loop that connects the N-terminal B (contains the active site distal histidine (His 106)) and C helices. This loop (BC) could play a role in maintaining the structural integrity of the active site. To further explore the significance of these residues, R117A, R117D, D597A and D597R variants were created using site-directed mutagenesis and expressed for the separately isolated domains. A major phase of reactivation kinetics is absent for R117A, leading to a substantial decrease in the rate and extent of reactivation. This implicates the residue as having a role in orienting the BC interhelical loop on the N-terminal domain for proper interaction with the C-terminal domain. The R117D KatGN variant was reactivated by KatGC by a slow monophasic process. The peroxidase activity observed following reactivation was indistinguishable from that of reactivated KatGN. Conversely, reactivated R117D KatGN showed only 30% of the catalase activity of the unmodified KatGN. Spectroscopic measurements indicated a substantial increase in high-spin states consistent with the observed return of peroxidase activity in R117D KatGN. However, EPR measurements showed a distinct difference in the distribution of high-spin states in comparison to wild-type KatG and reactivated KatGN. In contrast to far-UV CD measurements for R117D KatGN, D597R KatGC showed a substantial disruption of secondary structural content. Consistent with this observation, D597R KatGC was unable to drive reactivation of KatGN or R117D KatGN. These results show important roles for R117 and D597 in catalase-peroxidase function, but confirm that this has less to do with their interaction with one another and is more attributed to their effects on the structure and function of the individual domains. The disruption of KatGC structure by the D597R substitution indicates the importance of the residue to the structural integrity of a normally robust domain. Moreover, the result suggests that while KatGC is able to reactivate multiple conformers of KatGN, neither KatGN nor its R117D variant is able to facilitate the refolding of D597R KatGC. We also investigated the strictly conserved Tyr111 (Y111) residue on the N-terminal domain BC loop and the strictly conserved Arg484 (R484) on the B’C’ loop of the C-terminal domain. Spectroscopic and kinetic evaluation revealed minimal changes in the catalase and peroxidase activities upon reactivation of the Y111A KatGN variant with KatGC. However, the kinetics of Y111A catalase recovery were considerably slower than all other features of reactivation, indicating that a feature unique to catalase activity was impaired by the substitution. On the other hand, a 10-fold decrease in catalase and a total loss of peroxidase activity was observed for the reactivation of KatGN and Y111A KatGN with the R484A variant. These results suggest that R484 may play a role in supporting the B’C’ loop structure. This loop may be essential for proper interaction with the N-terminal domain’s BC loop, which in turn plays a major role in maintaining proper active site architecture for KatG function.