Membrane Pharmacy Structure Dynamics 

Research group : Priv.Doz. Dr. Thomas Nawroth 

Regulation of ATP-syntase   

General

ATP-Synthase and its catalytic fragment F1ATPase exhibit a high degree of molecular regulation in vivo and in vitro. If that would not be the case, for example a plant would consume all the ATP beeing synthesized by photosynthesis during the day in the following night. Some known regulative factors of ATP-synthase and F1ATPase are:
- the electrochemical proton potential difference of the membrane, also depicted as "membrane energization" (i.e. delta-p > 100 mV);
- the dissociation of an inhibitor protein (subunit epsilon in E. coli and chloroplasts, delta in Micrococcus luteus  (see reference F8, F18, F20), IF1 / AIP in mitochondria);
- the temperature T
- the reduction of an extra sulphur-bridge (Cys2) by Thioredoxin produced in the active photosynthesis chain in chloroplasts under illumination.
- the exchange or loss of nucleotide from one of the three non-catalytic nucleotide centers
- the concentration dependent load of nucleotides (ADP, ATP, GTP, GDP, nothing) into the three catalytic nucleotide centers, e.g. shifting the reaction form the slow single site catalysis over the dual site mechanism to the multisite catalysis.
For chloroplasts ATP-Synthase Strothmann (Düsseldorf) has detected > 40 native modifications (conformations) ! Such a complex regulation cannot be described by a simple R,T-scheme as suitable for oxygen transport proteins (Haemoglobin etc.). Additionally the enzyme may be regulated at the genetic level (expression).
The major part of the knowledge about the regulation of ATP-Synthase and F1ATPase from Micrococcus luteus is summarized in thesis T20. An overview is given in the two regulation schemes:

F1ATPase regulation for Micrococcus luteus

As shown in Fig.1 the F1ATPase from Micrococcus luteus ATCC4698 is at least regulated by:
- the dissociation of an inhibitor protein (subunit epsilon in Escherichia coli and chloroplasts, delta in Micrococcus luteus  (see reference F8, F18, F20), AIP in mitochondria);
- the temperature T (in two steps)
- the concentration dependent load of nucleotides (ADP, ATP, GTP, GDP, nothing) into the three catalytic nucleotide centers, e.g. shifting the reaction form the slow single site catalysis over the dual site mechanism to the multisite catalysis (not shown in the scheme).
The kinetics of the conversions and the activation energies and entropies have been estimated (T20, T14). A kinetic model fits the experiments in the temperature interval 10-70°C by 95%. F1L is the conformation of the enzyme in a stock solution in the refrigerator (< 30°C); F1H(-delta) is the modification after incubation at low concentration (< 10 mg/l) at high temperature (37°C) for 90 min., i.e. in the usual ATP hydrolysis test. Any conditions in and between refer to inhomogenous mixtures of several modifications !!!  The most important result for time resolved studies with this enzyme is the extraordinaryly high activation enthalpy dH = 205 kJ/mol for the hydrolysis of CaATP of the low temperature form of the complete F1ATPase modification F1L. This is the key for time resolved investigations, because it allows to slow down the reaction speed by three orders from 4 ms (37°C) to the second scale.
 
Fig.1: The changes of structure and function of F1ATPase from Micrococcus luteus correspond to the dissociation of the inhibitory delta-subunit (reactions 2, 3), the reversible tempereature dependence (reactions 1, 4, 5) and to irreversible thermical denaturation (reactions 6, 7). Below the threshold temperatures Ts1 and Ts2 no significant denaturation is observed. This F1ATPase exists at lest in 5 enzymatically active modifications (conformations) (from T20, reference F8).

ATP-synthase regulation for Micrococcus luteus

As shown in Fig.2 the ATP-synthase from Micrococcus luteus ATCC4698 (see thesis T15, T6, T17, T19, references F16, F17, F18, F20) is at least regulated by:
- the electrochemical proton potential difference of the membrane, also depicted as "membrane energization" (i.e. delta-p > 100 mV);
- the dissociation of an inhibitor protein (subunit epsilon in Escherichia coli and chloroplasts, delta in Micrococcus luteus  (see reference F8, F18, F20), IF1 / AIP in mitochondria);
- the temperature T  (not shown in the scheme)
- the concentration dependent load of nucleotides (ADP, ATP, GTP, GDP, nothing) into the three catalytic nucleotide centers, e.g. shifting the reaction form the slow  single site catalysis over the dual site mechanism to the multisite catalysis (not shown in the scheme)
The regulation resembles that found for ATP-synthase from mitochondria (see review of P.L. Pedersen). If one takes into account the temperature dependence (2 state levels) and the nucleotide dependence (3 state levels), not shown in the scheme, one has to expect about 24 possible active enzyme modifications (4 x 2 x 3) - a nice example how nature adapts thermodynamic yield to varying life conditions.
 
Fig.2: The changes of structure and function of ATP-synthase from Micrococcus luteus corresponds at a given temperature and ATP/ADP-level to the membrane energization delta-p and the dissociation of the inhibitory delta-subunit (in E. coli : epsilon) (from T20).

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email to: nawroth@MPSD.de   update : 15.10.2013