NorthPark University1, Physics, Chicago, IL 60625 University of Nebraska at LIncoln2, BioChemistry, Lincoln, NE 68501
Abstract: Proline utilization (PutA) from E. Coli is a trifunctional flavoenzyme that catalyzes the conversion of proline to glutamate. PutA also has the ability to bind DNA, in its oxidized state (FAD), acting as an autogenously transcriptional repressor and conversely when reduced (FADH2) it peripherally binds the inner membrane of E. coli where it relays electrons from the oxidation of proline to the electron transport chain. Residues 3-46 are responsible for the DNA binding and the major membrane binding domain is thought to be somewhere on the c-terminal end of the protein. In the domain (PRODH) which catalyzes the oxidation of proline to pyrroline-5-carboxylate, a small patch of arginine residues (423, 425, 426, and 427) exists on the surface of the protein that forms a net positive charge under physiological conditions. Details about how arginine 425 and 427 are important for the function of PutA are the goal of this study. This small patch of arginine residues could be involved in the protein’s ability to bind DNA and membrane. After conducting gel shift mobility assays, it was shown that EcPutA R425A and R427A have higher dissociation constants, Kd, as compared to the wild type protein. The Kd for R425A and R427A are 254 and 333.7 nM, respectively, where the published Kd of the wild type protein is roughly 45nM. The PutA’s ability to bind proline was tested by finding the Michaelis-Menten equilibrium constant (Km). The turn-over rate, Kcat, for R425A and R427A is 24 and 18.3 s-1 respectively. The protein’s ability to become fully reduced is similar to the wild type protein, and the functional membrane binding activity for these mutants is still being studied.
[Abstract (DOC)]