Adenylate kinase thermodynamics

Backmann, J. Schafer, G. Wyns, L. Bonisch, H. Thermodynamics and kinetics of unfolding of the thermostable trimeric adenylate kinase from the archaeon Sulfolobus acidocaldarius. J. Mol. Biol., 284, 817-833 (1998)... 

After these general comments let us further test the idea of thermodynamic buffering on an experimental basis by repeating the above experiment but this time in the presence of an inhibitor of adenylate kinase, namely, diadenosine pentaphosphate. As is depicted in Fig. 6b the buffering effect of the adenylate kinase is abolished by inhibiting this enzyme and it becomes now possible to drive the system beyond the state of optimal efficiency by increasing the hexokinase concentration in the medium. Note that it was not possible to measure points closer to level flow than the ones shown in the figure. This is due to technical reasons. At the lowest phosphate potentials the ATP/ADP ratios where of the order... 

In order to obtain a more intuitive insight into the mechanism of thermodynamic buffering we calculated the effects of thermodynamic buffering on the entropy production of the system. The entropy production of oxidative phosphorylation with an attached load is given in equation (8). A convenient way to introduce the contribution of the adenylate kinase reaction to this system is to consider L/ as an overall load conductance embracing the effects of the adenylate kinase reaction as well as the effects of the true extrinsic load conductance of the irreversible ATP utilizing... 

All of the effects of the catecholamines bound to (3 adrenergic receptors and of glucagon, ACTH, and many other hormones appear to be mediated by adenylate cyclase. This integral membrane protein catalyzes the formation of cAMP from ATP (Eq. 11-8, step a). The reaction, whose mechanism is considered in Chapter 12, also produces inorganic pyrophosphate. The released cAMP acts as the second messenger and diffuses rapidly throughout the cell to activate the cAMP-dependent protein kinases and thereby to stimulate phosphorylation of a selected group of proteins (Fig. 11-4). Subsequent relaxation to a low level of cytosolic cAMP is accomplished by hydrolysis of the cAMP by a phosphodiesterase (Eq. 11-8, step fr).166/167 jn thg absence of phosphodiesterase cAMP is extremely stable kinetically. However, it is thermodynamically unstable with respect to hydrolysis. 

Hayaishi and colleagues, who devised the purification for the Brevibacter-ium liquefaciens enzyme, used it to characterize the reversibility of the adenylate cyclase reaction (Kurashina et ai, 1974) and found that the equilibrium constant for the reaction written in the direction of cyclic AMP formation is 0.12 Mat pH 7.3 at this pH the rates of the forward and reverse reactions are comparable but about the rate of the forward reaction measured at its pH optimum, pH 9. Our plan for determining the stereochemical course of the reaction is shown in Fig. 14. Since we had synthesized the diastereomers of cyclic [, 0]dAMP, we would use the cyclase to catalyze their pyrophosphorolysis and form the diastereomers of [a- 0, 0]dATP. However, the thermodynamics of the cyclase reaction prevents an efficient conversion of cyclic dAMP to dATP, so this reaction was coupled to the glycerol kinase reaction the kinase reaction utilizes the thermodynamic instability of the )J,y-anhydride bond to displace the overall equilibrium to favor the synthesis of the diastereomers of [a- 0, 0]dADP. Both the cyclase and glycerol kinase can utilize deoxyadenosine nucleotides as substrates, but only the cyclase reaction can alter the configuration of the chiral phosphorus atoms. 

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