Adenosine kinase substrate specificity

Table 2.2. SUBSTRATE SPECIFICITY OF ADENOSINE KINASE A. Variation of substituents on purine ring... 

The substrate specificities of both mammalian and yeast hexo-kinases have been extensively studied (76,77). Nevertheless, work in this area continues both in the search for isoenzyme specific inhibitors and in increasingly detailed investigations of the catalytic mechanism. Recently potential transition state analogs PI-(adenosine-5 )-P3-glucose-6 triphosphate (Ap -glucose) and P1-(adenosine-5 )-P4-glucose-6 triphosphate (Ap.-giucose) were tested as inhibitors of four hexokinase isoenzymes. However, they were found to exhibit less affinity for the enzyme than either of the natural substrates alone (78). 

Leishmania adenylosuccinate synthetase has a narrow substrate specificity but accepts several IMP analogs which include allopurinol ribonucleotide (34). The GMP reductase from L. donovani is quite different from the human GMP reductase (35) and IMP analogs are more potent inhibitors for it. Other leishmanial enzymes that have been investigated include IMP dehydrogenase (36), nucleoside hydrolase and phos-phorylase activities (37,38), adenosine kinase (39), nucleotidases (40) and the adenylosuccinate lyase (34). 

Miller, R. L., Adamczyk, D. L., Rideout, J. L. and Krenitsky, T. A. (1982) Purification, characterization, substrate and inhibitor specificity of adenosine kinase from several Eimeria species. Mol. Biochem. Parasitol. 6 209-223. 

Generally, all conversions in the biosynthetic direction, i.e. iPARMP— iPAR— iPA (catalysed by 5 -nucleotidase, (EC 3.1.3.5), and adenosine nucleosidase, (EC 3.2.2.7), respectively, c/. Fig. 2) may also proceed in the opposite direction, i.e. base-— nucleoside — nucleotide (catalysed by adenosine phosphorylase and adenosine kinase, respectively). All these enzymes require both Ade and iPA or Ado and iPAR, respectively, as substrates. They were characterised in wheat germ [15-18] and lupin seeds [19]. Interestingly, no K, -constants were reported for Z-type cytokinins (see summary in [22]). However, as seen in H-labelled Z-derivatives feeding experiments, Z-type cytokinins are also interconverted in a similar way [82,121,122]. Moreover, the specificity of these enzymes is not too strict with respect to the side chain configuration and one may speculate that this complex may function for most if not all native cytokinins [21,81]. 

Datta R, Das I, Sen B et al. Homology-model-guided site-specific mutagenesis reveals the mechanisms of substrate binding and product-regulation of adenosine kinase from Leishmania donovani. Biochem J 2006 394(Pt l) 35-42. 

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 adenosine kinases from animal cells have broad specificities with respect to their substrates. Thus, ATP, ITP, and GTP can all serve as phosphate donors, althoi h to varying degrees. The nucleoside triphosphate is probably involved as the Mg + complex, and the Mg +-nucleoside triphosphate ratio is critical. The Michaelis constant for ATP is about 5-10 X 10 M, depending on the Mg + concentration. The molecular weight of the rabbit liver enzyme is about 233,000. 

Although yeast adenosine kinase is relatively specific with respect to its purine nucleoside substrate (55), the enzymes from animal tissues phosphorylate a variety of nucleosides, in which either purine base or... 

Studies of purified adenosine kinases have made it clear that inosine and guanosine are not among the substrates of this enzyme, in spite of its broad specificity, and for many years it was not certain whether they were enzymatically phosphorylated or not. Recently, however, LePage and his co-workers 59, 60) have provided data supporting the existence of separate inosine and guanosine kinases in crude extracts of mouse tumors, but these activities have not yet been purified. 

Regardless of the physiological role of the purine nucleoside kinases, adenosine kinase is very important in cancer chemotherapy with purine nucleoside analogues. Attempts are being made to treat tumor cells with purine nucleosides in which the base and sugar moieties have been altered from the normal structures. Conversion to nucleotides is necessary for the expression of the pharmacological activity of many of these compounds, and this is accomplished through the broad substrate specificity of adenosine kinase. 

In different tissues, for reasons that we do not understand, administration of 3-DZA can produce either the inhibition of the hydrolase, with the resulting increase in the intracellular concentration of AdoHcy, or its utilization as a substrate with the formation of 3-DZA-Hcy. In most tissues both effects can be observed. 3-DZA, like adenosine, is readily taken up by the cell through facilitated diffusion, and is extremely specific in its mode of action. As mentioned above, 3-DZA is neither a substrate nor an inhibitor for adenosine deaminase and adenosine kinase. It is, therefore, not incorporated in the acid soluble nucleotide pool and hence is not... 

T. A. Krenitsky and G. B. Elion, Adenosine kinase from rabbit liver. II. Substrate and inhibitor specificity, J. Biol. 

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. 

Animal and bacterial enzymes that utilize or synthesize carbamyl phosphate have activity with acetyl phosphate. Acyl phosphatase hydrolyzes both substrates, and maybe involved in the specific dynamic action of proteins. Ornithine and aspartic transcarbamylases also synthesize acetylornithine and acetyl aspartate. Finally, bacterial carbamate kinase and animal carbamyl phosphate synthetase utilize acetyl phosphate as well as carbamyl phosphate in the synthesis of adenosine triphosphate. The synthesis of acetyl phosphate and of formyl phosphate by carbamyl phosphate synthetases is described. The mechanism of carbon dioxide activation by animal carbamyl phosphate synthetase is reviewed on the basis of the findings concerning acetate and formate activation. 

In order to enhance affinity and selectivity for Brc-Abl, we modified the inhibitor methylating at positions I and II (Fig. 7.5d). The synthesis of the wrapping prototype recapitulates imatinib synthesis [38], as described in [39], To test whether the specificity and affinity for Brc-Abl improved, we conducted a spectrophotometric kinetic assay to measure the phosphorylation rate of peptide substrates in the presence of the kinase inhibitor at different concentrations. This assay couples production of adenosine diphosphate (ADP), the byproduct of downstream phosphorylation, with the concurrent detectable oxidation of reduced nicotinamide adenosine dinucleotide (NADH). The oxidation results upon transfer of phosphate from PEP (phospho-enolpyruvate) to ADP followed by the NADH-mediated reduction of PEP to lactate. Thus, phosphorylation activity is monitored by the decrease in 340 nm absorbance due to the oxidative conversion NADH->-NAD+ [34, 39]. 

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. 

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