ATP kinases

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

Although most of the conserved phosphate-binding residues in the ATP-binding site are crucial for the catalytic process, they do not contribute much to the free energy of binding of the ATP kinase, which is nicely reflected in the equipotent affinity of PKA for ATP, ADP, and adenosine [10],... 

NMR method in this case 15N-HSQC, depends on following the movement of cross-peaks as a small molecule is added. If a titration is performed, an NMR-XD can be extracted, as shown in the graph, (b) Ligand-detected STD NMR method (i) ID control spectrum of AMP/kinase (ii) STD spectrum of AMP/kinase only resonances of atoms that contact the protein are present in the STD spectrum (Hi) STD spectrum of ATP/kinase complex (iv) STD of ATP/kinase/competitor, the STD signal due to ATP is decreased because ATP is partially displaced from the binding site by the competitor, and new STD signals for the competitor appear, compared to spectrum (Hi)... 

Hexokinases, other kinases, and many other enzymes that catalyze reactions involving the hydrolysis of ATP require Mg. The Mg forms a complex with the phosphate groups of ATP. Kinases also require K+. 

Fig. 10. Array-based phosphorylation assay using an exogenous kinase. An array of 96 human protein kinases was printed in dupiicate and assayed in the presence and absence of exogenous FES kinase, (a) Schematic of assay, (b) 100 p.M ATP pius kinase buffer oniy. (c) 100 p,M ATP, kinase buffer pius exogenous FES kinase. The assays were deveioped using a fiuorescentiy iabeiied anti-phosphotyrosine antibody and reveaied a number of substrates for FES kinase, including MKNK1, STK6, and STK25, as marked.

The majority of receptors affecting acid secretion belong to the G7 (seven transmembrane segments) class of receptors (H2, muscarinic, CCK2, somatostatin, prostaglandin). The EGF/TGF-a receptor, which also inhibits acid secretion, is a tyrosine kinase receptor (single transmembrane segment with an intracellular ATP kinase domain). 

Calculation of Conformational Free Energies for a Model of a Bilobal Enzyme Protein kinases catalyze the transfer of phosphate from adenosine triphosphate (ATP) to protein substrates and are regulatory elements of most known pathways of signal transduction. 

The catalytic subunit of cAPK contains two domains connected by a peptide linker. ATP binds in a deep cleft between the two domains. Presently, crystal structures showed cAPK in three different conformations, (1) in a closed conformation in the ternary complex with ATP or other tight-binding ligands and a peptide inhibitor PKI(5-24), (2) in an intermediate conformation in the binary complex with adenosine, and (3) in an open conformation in the binary complex of mammalian cAPK with PKI(5-24). Fig.l shows a superposition of the three protein kinase configurations to visualize the type of conformational movement. 

Fig. 2. Conformational free energy of closed, intermediate and open protein kinase conformations. cAPK indicates the unbound form of cAMP-dependent protein kinase, cAPKiATP the binary complex of cAPK with ATP, cAPKiPKP the binary complex of cAPK with the peptide inhibitor PKI(5-24), and cAPK PKI ATP the ternary complex of cAPK with ATP and PKI(5-24). Shown are averaged values for the three crystal structures lATP.pdb, ICDKA.pdb, and ICDKB.pdb. All values have been normalized with respect to the free energy of the closed conformations.

Left side of Fig. 4 shows a ribbon model of the catalytic (C-) subunit of the mammalian cAMP-dependent protein kinase. This was the first protein kinase whose structure was determined [35]. Figure 4 includes also a ribbon model of the peptide substrate, and ATP (stick representation) with two manganese ions (CPK representation). All kinetic evidence is consistent with a preferred ordered mechanism of catalysis with ATP binding proceeding substrate binding. 

The biological transformations that involve ATP are both numerous and funda mental They include for example many phosphorylation reactions m which ATP trans fers one of its phosphate units to the —OH of another molecule These phosphoryla tions are catalyzed by enzymes called kinases An example is the first step m the metabolism of glucose... 

Kinases (Section 28 3) Enzymes that catalyze the transfer of phosphate from ATP to some other molecule Kinetically controlled reaction (Section 10 10) Reaction in which the major product is the one that is formed at the fastest rate... 

Adenosine is formed from ATP via a phosphatase cascade that sequentially involves the diphosphate, ADP, and the monophosphate, AMP. The actions of adenosine are terminated by uptake and rephosphorylation via adenosine kinase to AMP or by cataboHsm via adenosine deaminase to inosine and hypoxanthine. 

Ara-A is phosphorylated in mammalian cells to ara-AMP by adenosine kinase and deoxycytidine kinase. Further phosphorylation to the di- and triphosphates, ara-ADP and ara-ATP, also occurs. In HSV-1 infected cells, ara-A also is converted to ara-ATP. Levels of ara-ATP correlate directly with HSV rephcation. It has recently been suggested that ara-A also may exhibit an antiviral effect against adenovims by inhibiting polyadenylation of viral messenger RNA (mRNA), which may then inhibit the proper transport of the viral mRNA from the cell nucleus. 

The second structure, adenylate kinase (Figure 4.14b), has two such posi-I tions, one on each side of p strand 1. The connection from strand 1 to strand 12 goes to the right, whereas the connection from the flanking strands 3 and 4 both go to the left. Crevices are formed between p strands 1 and 3 and [between strands 1 and 4. One of these crevices forms part of an AMP-binding [site, and the other crevice forms part of an ATP-binding site that catalyzes the Iformation of ADP from AMP and ATP. 

Kinases (Section 28.3) Enzymes that catalyze the transfer of phosphate from ATP to some other molecule. 

FIGURE 3.13 Phosphoenolpyruvate (PEP) is produced by the euolase reaction (hi glycolysis see Chapter 19) and hi turn drives the phosphorylation of ADP to form ATP in the pyruvate kinase reaction. 

An example of a random, single-displacement mechanism is seen in the enzyme creatine kinase, a phosphoryl-transfer enzyme that uses ATP as a phosphoryl... 

Cyclic AMP-dependent protein kinase is shown complexed with a pseudosubstrate peptide (red). This complex also includes ATP (yellow) and two Mn ions (violet) bound at the active site. 

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