ATP sites

Deluca, M., and McElroy, W. D. (1984). Two kinetically distinguishable ATP sites in firefly luciferase. Biochem. Biophys. Res. Commun. 123 764-770. 

The kinetically deduced existence of two classes of substrate sites may also account for the molar ratio between ATP analogs and inhibitors on the one hand and phosphoenzyme on the other hand. This ratio has been reported to be 2 1 for the ATP analogs adenylyl imido diphosphate (AMP-PNP) [135] and 2, 3 -0-(2,4,6-trinitrophenylcyclohexadienylidine)-ATP (TNP-ATP) [97], and also 2 1 for the ATP-site directed fluorescent inhibitors eosin [99] and FITC [49,50] and the transition-state inhibitor vanadate [126]. 

Fig. 8.1 Hypothetical two-dimensional model of human P-glycoprotein. Small circles amino acid residues large circles ATP sites squiggly lines N-linked glycosylation sites (modified from [15]).

Fig. 6.15 FAC-MS chromatograms of dual indicators for protein kinase Ca [32]. (a) In the chromatograms, the red lines correspond to a void marker, the blue lines correspond to the substrate-site indicator chelerythrine chloride and the magenta lines correspond to the ATP-site indicator PDl53035. Arrows...

The receptor relevance of BCUT descriptors has inspired several groups to apply them in conjunction with other methods. Beno and Mason reported the use of simulated annealing to optimize library design using BCUT chemistry space and four-point pharmacophores concurrently (33) and the use of chemistry spaces in conjunction with property profiles (52). The application of such composite methods to target class library design is readily apparent. Pirard and Pickett reported the application of the chemometric method, partial least squares discriminant analysis, with BCUT descriptors to successfully classify ATP-site-directed kinase inhibitors active against five different protein kinases... 

Exploration of Bulk Tolerance at ATP Sites. Non-covalent type inhibitors have also been used to study bulk tolerance around the ATP binding sites. In this vein Hampton and co-workers have both synthesized and tested as inhibitors a large number of adenine nucleotide analogs (Figure 2f) to probe the bulk tolerance at a number of positions on the parent compound (28-31) These compounds have been used to study systematically the isoenzyme selectivity of adenylate kinases, hexokinases, thymidine kinases and pyruvate kinases with respect to bulk tolerance at many sites on the ATP molecule. Some of the most isoenzyme specific results were obtained with pyruvate kinase isoenzymes K,L and M using ADP derivatives. Here 3 -0Me-ADP was found to inhibit pyruvate kinase preferentially with a ratio of inhibitory potency of 7.6 6.0 1.0 for the K,M and L isoenzymes, respectively. Another compound, 8-NHEt-ADP, was selective for the M isoenzyme, giving a ratio of 7.1 1.2 1.0 for the M, K and L forms, respectively. 

Affinity Labeling of Catalytic ATP Sites. Residues involved in ATP binding are potentially revealed by the use of affinity labels that are based on ATP s structure. Perhaps the most systematically studied of these compounds is 5 -fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been reported to label at least six kinases (32-A1). In the case of rabbit muscle pyruvate kinase such work has Indicated the presence of a tyrosine residue within the metal nucleotide binding site and an essential cysteine residue located at or near the free metal binding site (32). A similar reagent, 5 -FSBGuanosine, revealed the presence of two cysteine residues at the catalytic site of this same enzyme, both distinct residues from those modified by 5 -FSBA (33,34). With yeast pyruvate kinase both tyrosine and cysteine residues were modified by 5 -FSBA at the catalytic site ( ), and with porcine cAMP-dependent protein kinase a lysine residue was labeled at the active site (36). 

Affinity labeling ATP sites, 194-96 creatine kinase, 200-205 Amastatin, 94-96 Amide bond hydrolysis, 227 Amino acid sequences, renin inhibitors, 139,141f D-Amlno acids and activity,... 

ATP Site A Pyri(mi)dine-5-MeniberHet ocycle 4 Phos, NE. 

Fig. 14.5. Annotation for the ATP site inhibitor, SB203580. The core is Pyri(mi)dine-5-MemberHeterocycle 4, which is a hydrogen bond acceptor with the hinge region. The compound probes the Phosphate (Phos) and NE sites.

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