Enzyme Kinetics of H+-ATP Synthase of Chloroplasts
Date Issued
2016
Date
2016
Author(s)
Liu, Huei-Ling
Abstract
H+-ATP synthase is an integral membrane enzyme, which catalyzes ATP synthesis in bacteria, chloroplasts and mitochondria. In chloroplasts of spinach, ATP synthesis can be driven by proton concentration difference pH, and electric potential across the membrane. Our research could be divided into two distinct topics. One is the study for pH-dependent activity behavior of CFoF1, and the other one is the study for the critical-like behavior of CFoF1. To elucidate the pH-dependent activity behavior of CFoF1, we proposed a molecular kinetic model consisting of two proton relay groups, each being characterized with a specific proton dissociation constant, pK. From appropriately designed experiments, the pK values of the relay groups were extracted to be 5.1 for pKin and 8.3 for pKout, respectively. By aligning the subunit a of spinach with that of E.coli., we identified the candidate residues that may play the role of the proton relay groups: aAsp-197 and aGlu-198 for input relay groups, and aGlu-178, aAsp-179 and aLys-182 for output. We adapted the quasichemical theory of solvation to calculate the theoretical pK values of the candidate residues, and identified the most plausible candidates of the proton relay groups. Overall, a molecular kinetic mechanism for protons conducting through CFoF1 during ATP synthesis and its physical implications on the specific reactivity behavior of CFoF1 are presented. To observe the critical-like behavior of CFoF1, we performed the activity experiments with two factors, temperature and calcium ion, respectively, which were reported to be capable of inducing critical-like behavior of lipid bilayers. We found that at around 40℃, the activity of CFoF1, reconstituted in PC/PA 19:1 liposomes critically increased 2 times of the room temperature one. To observe the Ca++ effect, we prepared the CFoF1-reconsituted liposomes with various internal Ca++ concentrations to measure the activity of ATP synthesis. We found that at around [Ca++] = 2.5 mM, the activity seemed to have critical increase, and this Ca++ concentration happened to be located at the critical-like region of the pure liposomes. To explain our experimental observation with critical fluctuation, we establish a generalized Langevin equation, which is incorporated with the critical correlation time, to simulate the rotational movement of CFo. We found that with long correlation time, the rotational frequency increased 2.3-4.5 times of the one with short correlation time. This indicates that the critical fluctuation of the membrane could enhance the rotational movement of CFo.
Subjects
H+-ATP synthase
proton relay groups
pH dependency
chloroplasts
critical behavior
Type
thesis
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