Submitted to: Archives Of Biochemistry and Biophysics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/18/2004
Publication Date: 2/2/2005
Citation: Tovar-Mendez, A., Hirani, T., Miernyk, J.A., Randall, D.D. 2005. Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase. Archives Of Biochemistry and Biophysics. 434(1):159-168. Interpretive Summary: Respiration is the use of energy by living cells to do work. Both growth and reproductive success are directly coupled to rates of respiration. As a result, respiration must be carefully controlled, or wasted energy would cause decreased crop yields and reduced agricultural productivity. The control of respiration in plant cells is a subject of ongoing study. The activity of a protein thought to be important in overall control of respiration is itself controlled by a different, associated protein. A method was developed that allows study of the controlling protein in the laboratory. The chemical components that make up the controlling protein were modified to define which contributes the most to overall control of respiration. Based upon these comparisons, a mechanism of action for the protein was predicted which will serve as the target for future tests of this prediction. This information will be important to researchers in their attempts to increase agricultural productivity by altering the control of plant cell respiration, and to other plant scientists who will try to design more efficient crop plants through either classical breeding or biotechnology.
Technical Abstract: In the previously proposed mechanistic model for pyruvate dehydrogenase kinase (PDK), "Glu238" within the N-box acts as a base catalyst that is polarized by an adjacent His residue. However, the "Glu polarizing His" is absent in plant PDK sequences, leading us to reexamine the model. The pH dependence of kcat of Arabidopsis thaliana PDK indicates ionizable groups with pK values of 6.2 and 8.4 are needed for catalysis, and the temperature dependence of these values suggests the acidic pK could be due to a carboxyl- or imidazole-group. The E238A, E238Q, E238H, and K241 AtPDK mutants all had residual activity and elevated Km ATP values. The acidic pK value of the E238A mutant was shifted to 5.5, while the values of the K241A mutant were unchanged. The kcat values for the E238Q and E238H mutants were pH independent. Finally, the H233A, L234H, and L234A mutants had the same pKs values as wild type PDK, which does not support the proposal of a "Glu-polarizing" His. These results led us to counter that conserved Glu, Lys, and Asn residues together act to coordinate Mg2+ in a position that allows productive ATP binding. The conserved Lys residue might polarize the gamma-phosphate of ATP and/or stabilize a reaction intermediate. The Glu238 residue could still act as a base catalyst, or alternatively, it could be critical in supplying a necessary acidic function. In the latter case, the base catalyst could be located on the PDK substrate, pyruvate dehydrogenase, or within the ATP-lid structure of PDK itself.