Affinity Labeling of Nucleotide-Binding Sites

Mitsuo Tagaya, Katsuyuki Tanizawa, and Toshio Fukui The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, Japan 

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Figure 5-7. Affinity labelling of nucleotide bind- hyde analogue. These groups react with accessible ing proteins. Nucleotides bound to protein are lysine residues in the region ofthe nucleotide bind-treated with periodate, which cleaves the vicinal ing site to form a Schiffbase adduct this can be diol groups to generate a highly reactive dialde- stabilised by reduction with NaHB4.

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). 

If the photoaffinity reagent has been designed well and there are a measurable number of tight binding sites for it, the protection experiment will usually succeed. Occasionally it will not for example, four polypeptides of sarcoma cells were labeled with low concentrations of 8-N3-CAMP but only three of the sites could be protected by cAMP. Presumably, the fourth site is not part of a cAMP binding protein but a different nucleotide binding site that just happens to have affinity for the reagent but not for cAMP. 

The title compound (I) can be viewed as an enzyme affinity label produced by attachment of a relatively small leaving group (chlorine) to a hydrophilic substrate (inosine 5 -phosphate) in the expectation that the enzymic substrate binding sites will contain a preponderance of hydrophilic and hence potentially derivatizable amino acid residues. Evidence indicates that (I) forms covalent bonds at the nucleotide binding sites of two of four purine nucleotide utilizing enzymes which were examined. 

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