Adenylate kinase substrate specificity

In a number of cases there may be a contaminating enzyme present which acts on one or more of the substrates, products, or effectors of the system under study. It may be necessary to include in the reaction mixture a specific inhibitor for that contaminating activity. For example, adenylate kinase is often present in preparations of a number of phosphotransferases. It is often advantageous, in such instances, to include a specific inhibitor of adenylate kinase (e.g., P, P -di(adenosine-5 )-tetraphosphate or P P -di(adenosine-5 )-pentapho-sphate). If an inhibitor of the contaminating activity is added as an additional constituent of the reaction mixture, the investigator should demonstrate that the inhibitor is not an effector of the enzyme under study. 

Nucleoside diphosphates (NDP) are synthesized from the corresponding nucleoside monophosphates (NMP) by base-specific nucleoside monophosphate kinases (Figure 22.9). [Note These kinases do not discriminate between ribose or deoxyribose in the substrate.] ATP is generally the source of the transferred phosphate, because it is present in higher concentrations than the other nucleoside triphosphates. Adenylate kinase is particularly active in liver and muscle, where the turnover of energy from ATP is high. Its function is to maintain an equilibrium among AMP, ADP, and ATP. Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase—an enzyme that, unlike the monophosphate kinases, has broad specificity. 

It was found that the enzyme is specific for (/ )-ATPaS but does not react with (S)-ATPaS. As shown in Scheme 43, when (/ )-ATPatS and l70-acetate are used as substrates, the 170 from acetate will be incorporated into the pro-5 position of AMPS if the reaction proceeds with retention of configuration or into the pro-/ position if inversion occurs. To determine the configuration of the 170-labeled AMPS (compound type 4), it is converted to (S)-ATPoS by stereospecific phosphorylation at the pro-/ oxygen catalyzed by adenylate kinase, followed by a second phosphorylation catalyzed by pyruvate kinase (144,145). By such a conversion, 170 should be incorporated into the nonbridging position of (5)-ATPaS if the step of acetate activation proceeds with retention of configuration. On the other hand, 170 should be located at the P—O—P bridging... 

Co(lII), or Rh(lII), which form inert complexes with nucleotides that exchange ligands on the time scale of days or weeks (especially at low temperatures). It is possible, for example, to separate the A and A isomers of CrATP and use them as substrates in single turnover experiments with various enzymes 23, 24). When the enzyme catalyzes multiple turnovers, the developing circular dichroic (CD) spectrum as one isomer is converted to a product without a CD spectrum can determine the screw-sense specificity. Thus, hexokinase and glycerokinase use the A isomer of CrATP as a substrate, and pyruvate kinase and myokinase (adenylate kinase) use the A isomer (25). The absolute configurations of the ADP and ATP complexes of these metal ions are now known and have been correlated with the CD spectra (26-30). 

Adenylate kinase, which in vivo catalyzes the equilibrium between adenosine mono-, di- and triphosphates, has been used extensively in the production of ATP (27, 41). Although the enzyme has a broad substrate specificity for nucleoside di- and triphosphates, the specificity for monophosphates is much more restrictive. Nonetheless, the specificity is... 

The first such enzymatic transphosphorylation to be studied was the formation of ADP from adenylate and ATP, catalyzed by an enzyme in rabbit muscle (S) this enzyme, myokinase, is one of a larger group of adenylate kinases. It is apparent today that the nucleoside monophosphate kinases constitute a large family of enzymes with different specificities for nucleotide substrates certain of these kinases have particular intracellular sites (see reference 4)-... 

Because the majority of the known nucleoside monophosphate kinase reactions require an adenosine phosphate as one of the substrates, they may be classified as those which (1) are specific for adenylate as a phosphoryl acceptor, and (2) as those which require ATP as a phosphoryl donor. (The adenylate kinase reaction obviously may be placed in either category.) There may exist an additional class of nucleoside monophosphate kinase reactions in which adenosine phosphates do not participate however, such enzyme activity has not been unequivocally demonstrated. 

Guanylate kinase has been partly purified from hog brain 10), from Escherichia coli 11), and from a transplantable mouse tumor, Sarcoma 180 12). These preparations show similarities with respect to substrate specificity and cation requirements, but differ with respect to pH optima and molecular weight. All three preparations are highly specific in that guanylate and deoxyguanylate are phosphorylated, but adenylate, ino-sinate, xanthylate, and cytidylate are not substrates. The brain and tumor enzymes also phosphorylate 8-azaguanylate. ATP or dATP are the specific phosphate donors for the three enzymes. 

Fractionation of extracts from calf thymus for kinase activity toward deoxyribonucleoside monophosphates has revealed the presence of at least four separate enzymes the kinase activities for dAMP, dGMP, dCMP, and dTMP are separate entities in this tissue 31). Each has been partly purified and examination of substrate specificities showed that the kinases for deoxyadenylate and deoxyguanylate also phosphorylate the ribosyl homologues, adenylate and guanylate, respectively. The deoxycytidylate kinase accepts as substrates both cytidylate and uridylate, but will not phosphor> late deoxyuridylate. The calf thymus thymidine monophosphate... 

UMP in a reaction that greatly resembles that of adenylate kinase. The reaction requires ATP and results in the formation of ADP and UDP. The complete purification of kinases of this type has not been achieved, but the partial separations already accomplished indicate that at least three, and possibly more enzymes with varying substrate specificities exist. These enzymes, studied in calf liver extracts, all use ATP as the phosphate donor, and show at least relative specificity for adenylic acid, uridylic acid, and cytidylic acid as phosphate acceptors. 

Recent studies on mitochondrial structure have demonstrated the localization of mitochondrial enzymes in two membranes and two potential spaces (Fig. 4). For a substrate to react with an enzyme located in the mitochondrial matrix, it must first penetrate both outer and inner membranes. The outer membrane does not appear to hinder the passage of small molecules and substrates. The inner mitochondrial membrane, more analogous to the lysosomal membrane, is relatively impermeable to small molecules and ions (Chappell and Crofts, 1966). For anionic substrates to reach the matrix dehydrogenases, specific transporting systems are present in the inner mitochondrial membrane. Even for diose enzymes located in the inner membrane, penetration of that structure must occur as the active center of such enzymes is only available from the matrix side of the membrane (Chappell, 1968). Such data demonstrate that the presence of a membrane does not in itself induce enzyme latency as creatinine kinase and adenylate kinase localized in the intermembrane space, do not show latency. Moreover, monoamine oxidase and NADH cytochrome c reductase, present in the outer mitochondrial membrane, do not show latency. 

The specific ATPase of liver mitochondria has been separated from adenylic kinase. This ATPase appears to be associated with minute particles and is activated by Mg++. ATP is the specific substrate and ADP serves as a potent inhibitor. The complete dephosphorylation of ATP by aged mitochondria is apparently the result of the combined action of the specific ATPase and adenylic kinases. 

The cAMP system also derives some of its specificity from the proteins that are substrates for cAMP-dependent protein kinase. It is an indirect regulatory system, since cAMP modulates the activity of cAMP-dependent protein kinase and the kinase, in turn, affects the activities of a variety of metabolic enzymes or other proteins. This indirectness is the basis for amplification of the hormonal signal. Activation of adenylate cyclase by binding of a hormone molecule to a receptor causes formation of many molecules of cAMP and allows activation of many molecules of the protein kinase, each of which in turn can phosphorylate many enzymes or other proteins. This amplification cascade accounts in part for the extreme sensitivity of metabolic responses to small changes in hormone concentrations. Finally, the response to increased cAMP concentrations within a cell usually involves several metabolic pathways and a variety of enzymes or other proteins. 

As might be noted in the preceding description, the specificity of neurotransmitter effects that are mediated by cAMP is determined at three basic levels. First of all, the nature of the neurotransmitter receptor will determine whether membrane-bound adenylate cyclase will be stimulated, inhibited, or left unaffected. Second, the nature, concentration, and intrinsic activity of the protein kinase and the phosphoprotein phosphatase will determine the pattern and degree of protein phosphorylation. Last, the identity and availability of the protein substrates will determine the ultimate biochemical consequences of cAMP elevations. 

Nelson and Carter 18) have purified thymidylate kinase about 5,000-fold from E. coli B and have shown that thymidylate was phosphorylated about seven times faster than deoxyuridylate. Uridylate was not a substrate, nor were adenylate, guanylate, cytidylate, or their deoxyribosyl counterparts. This enzyme was less specific for the triphosphate phosphoryl donor than the kinases previously discussed, because CTP, GTP, and the corresponding deoxyribonucleotides also served as phosphate donors, although less weU than ATP. 

Nucleoside monophosphate kinases have been isolated which are specific for adenylate as the phosphoryl acceptor, but will accept various nucleoside triphosphates as phosphoryl donors. Such an enzyme activity was partly purified from hog kidney by Gibson et at. 20). Heppel et al. 21) have separated a phosphokinase activity from calf liver which accepts only adenylate as the nucleoside monophosphate substrate, but uses ATP, GTP, CTP, UTP, or ITP as the phosphate donor. 

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