Adenylate kinase reaction mechanism

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... 

Weiss and coworkers pioneered in using single-molecule FRET to study the conformational dynamics and reaction mechanism of staphylococcal nuclease.85 Lu and coworkers used it to study the conformational dynamics of T4 lysozyme during catalysis.58 Hammes, Benkovic, and coworkers studied dihydrofolate reductase,25 the enzymes involved in T4 primosome86 and replisome.87 88 Yang and coworkers studied adenylate kinase.89 Here, we use the T4 lysozyme study to illustrate the approach.5... 

These comparative studies constituted the first example of an enzyme-catalyzed hydrolysis reaction whose stereochemical course was unaffected by sulfur substitution. At the time these experiments were performed, the stereochemical courses of the reactions catalyzed by glycerol kinase (83, 84) and by the bacterial adenylate cyclase (85, 86) had already been compared in the laboratories of Knowles and Gerlt, respectively, and these were also found to be unaffected by the sulfur substitution. A number of other comparisons of this type have been made, and in no case were the stereochemical consequences of the reactions studied with chiral phosphate esters and the chiral thiophosphate analogs found to differ. This agreement suggests that the necessary use of oxygen chiral thiophosphate monoesters to study the stereochemical course of phospho-monoesterases will provide pertinent results for ascertaining whether phosphory-lated intermediates are involved in the reaction mechanism. 

The configuration about P is inverted in this reaction, which must, therefore, proceed by a mechanism involving an uneven number of displacements at P, most likely one in this case. Adenylate kinase catalyzes reaction (6), a [ 0] thiophospho transfer from (/ p)-[7- 02]ATPyS to AMP to form (5p)-[/3- 0] ADP S (11). 

Again, the configuration is inverted. Nucleoside diphosphate kinase catalyzes the same transfer, but to a nucleoside diphosphate rather than to AMP, and with retention of configuration rather than with inversion (70). The mechanism of action of adenylate kinase involves a single displacement at P and that of nucleoside diphosphate kinase involves a double displacement at P via an intermediate phosphoenzyme. Although alkaline phosphatase is not classified as a phosphotransferase, it catalyzes transphosphorylation via the same phosphoenzyme that is the intermediate in the phosphatase reaction. This enzyme catalyzes reaction... 

Acyl adenylate intermediates seem the general rule for acyl activation, but alternate mechanisms are known. An example is the succinyl thio-kinase reaction. The mammalian enzyme system utilizes guanosine triphosphate (GTP) or inosine triphosphate (ITP), although similar ATP-requiring enzymes are known from plants and bacteria. In addition to the coenzyme A derivative, a nucleoside disphosphate and inorganic phosphate are produced. 

Recently, Rhoads and Lowenstein (6) have utilized initial velocity and equilibrium kinetics to study the kinetic mechanism of muscle adenylate kinase from rabbit muscle and have concluded that the reaction is of the random bi-bi type. Isotope exchange between adenylate and ADP is... 

As shown in Figure 10.8, phosphodiesterase catalyses the hydrolysis of cAMP to yield 5 -AMP, thus providing a mechanism for termination of the intracellular response to the hormone. Under normal conditions, 5 -AMP is then phosphorylated to ADP by the reaction of adenylate kinase — it is only under conditions of relatively low ATP... 

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. 

Hunzicker-Dunn et al. [73] and Ezra and Salomon [74] suggested that the desensitization of the ovarian adenylate cyclase system by LH involves a phosphorylation reaction. The latter may alter the conformation of the receptor and/or Gs. If phosphorylation is the mechanism it may well be mediated by protein kinase C because phorbol esters, which are presumed to activate this enzyme, mimic LH induced desensitization in Leydig tumour cells [75,76]. There are, however, differences between LH- and phorbol ester induced desensitization [76], Cyclic AMP-dependent protein kinase is probably not involved because dibutyryl cyclic AMP does not mimic LH induced desensitization in Leydig cells [70]. 

Mechanisms. cAMP plays a central role as a second messenger in the regulation of cell metabolism (14). Binding of cAMP with the regulatory subunit of a protein kinase initiates a cascade of enzymatic reactions that ultimately lead to the breakdown of glycogen and release of glucose to the blood stream. The intracellular concentration of cAMP represents a balance between the action of adenylate cyclase (which produces cAMP... 

The importance of the phosphoenzyme in the mechanism of action of succinyl-CoA synthetase in reactions (27a)-(27c) is also unknown. The mechanisms of action of aminoacyl-tRNA synthetases and of acyl-CoA synthetases do not include covalent enzymic intermediates. The fact that succinyl-CoA synthetase involves succinyl phosphate as the activated substrate, whereas the others involve acyl adenylates, does not explain the difference. There is no chemical catalytic basis for the mechanisms of the formation of these intermediates to vary in this way. Moreover, acetate kinase produces acetyl phosphate without the intermediate formation of a phosphoenzyme, so that at least acetate kinase has the capacity to catalyze direct phosphorylation of a carboxylate group. 

If adenosine (adenine riboside) is formed by either of the two mechanisms discussed above, the formation of adenylic acid (AMP) and the pyrophosphates depends upon the introduction of a phosphate group. Kinases for the formation of riboside 5 -phosphates from the riboside and ATP are known, Another reaction sequence (reaction b) has also been found for the formation of nucleotides (IV). Individual enzymes have been found for handling orotic acid (a pyrimidine precursor) and adenine... 

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