Phosphoryl transfer adenylate kinase

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted to their di- and triphosphates for polymerization to RNA, for reduction to 2 -deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate to adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside mono-phosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates by the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)... 

Phosphoryl group transfers from ATP result in an accumulation of ADP for example, when muscle is contracting vigorously, ADP accumulates and interferes with ATP-dependent contraction. During periods of intense demand for ATP, the cell lowers the ADP concentration, and at the same time acquires ATP, by the action of adenylate kinase ... 

Kinases Direct transfer of terminal phosphoryl group of ATP to substrate Creatine kinase Adenylate kinase Hexokinasc Phosphoglycerate kinase Pyruvate kinase Protein kinase Myokinase Phosphofructokinase Type 1 (M-S-E) Type 1 Type 1 Type 1 Type 2 (S-M-E)... 

The stereochemical consequence of [lsO]thiophosphoryl and [l60, nO, lsO]phos-phoryl group transfer catalyzed by seven phosphotransferases were simultaneously determined in the author s laboratory and in the laboratory of J.R. Knowles. The first to be completed was the demonstration of inversion by adenylate kinase however, prior to that glycerokinase, hexokinase and pyruvate kinase had been shown to catalyze [l80]thiophosphoryl group transfer with the same stereochemical consequences, either all with inversion or all with retention. Glycerokinase was later shown to catalyze both [l60, nO, l80]phosphoryl and [l80]thiophosphoryl group... 

Phosphoryl transfer is a fundamental reaction in biochemistry and is one that was discussed in mechanistic and structural detail earlier (Section 9.4). Kinases are enzymes that catalyze the transfer of a phosphoryl group from ATP to an acceptor. Hexokinase, then, catalyzes the transfer of a phosphoryl group from ATP to a variety of six-carbon sugars (hexoses), such as glucose and mannose. Hexokinase, like adenylate kinase (Section 9.4.2) and all other kinases,... 

A conceptually similar approach can be used to determine the configurations of chiral phosphate monoesters of chiral alcohols without the necessity for transferring the chiral phosphoryl group to 1,3-butanediol. For example, in a number of stereochemical studies of hydrolysis reactions performed in the author s laboratory, 3 - or 5 -nucleotides were obtained as reaction products. In the case of samples of 5 -AMP, these can be enzymically converted to isotopically labeled cyclic 3, 5 -nucleotides by initial pyrophosphorylation with adenylate kinase and pyruvate kinase, followed by enzymic cyclization with inversion of configuration by a bacterial adenylate cyclase (20) (see Figs. 6 and 7). In the case of samples of 3 -nucleotides or 5 -nucleotides other than 5 -AMP, the cyclization reactions can be accomplished chemically (23). In either case, following chemical con-... 

Other examples of crossover activity are known. ASA has been shown to possess cyclic phosphodiesterase activity. Adenylate kinase transfers the 7-phosphoryl group of ATP, but can also accept as a substrate the sulfuryl analogue 7-sulfiiryl-ADP. Sulfoenolpyruvate, the sulfate analogue of phosphoenolpyruvate, is a substrate for pyruvate kinase, producing pyruvate and adenosine-5 -sulfatopyrophosphate. Many other examples probably remain undiscovered. 

Of primary importance are enzymes which catalyse phosphate transfer or phosphorylation. These have been known variously as kinases, phosphotransferases phosphorylases, phosphokinases, trans-phosphorylases and so on. They show a wide range of molecular weights. Adenylate kinase, for example, has a molecular weight of 21,000, whereas yeast fructokinase has a molecular weight of 580,000. 

Biosynthesis of ATP. ATP is the irrunediate product of all cellular processes leading to the chemical storage of energy. It is biosynthesized by phosphorylation of ADP in the course of Substrate phosphorylation (see). Oxidative phosphorylation (see) and non-cyclic Photophosphorylation (see) in plants. Energy in the form of a third phosphate may also be transferred to ADP from other high-energy phosphates, such as creatine phosphate (see Creatine) or other nucleoside triphosphates, or in the adenylate kinase reaction. 

For adenylate kinase, P-NMR measurements yield A 0.4 and K gq 1.5 at 4 C and pH 7.0. The rate of phosphoryl transfer was estimated to be 600-700 s (Nt eswara Rao et ai, 1978a), nearly an order of magnitude faster than the rate of overall reaction. Once again the interconversion rates are not rate-limiting for the reaction. Computer simulations of the line shapes of some of the spectra in Fig. 10 and others obtained on the spectrum yield not only a reliable phosphoryl transfer rate of600 sr on the enzyme but also provide evidence for and allow an estirrrate of the rate of exchange of the two ADP molecules between the acceptor and donor sites on the enzyme (see Section IV for a brief description of these results). 

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