Affinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine.
Aldolase contains one tight binding site and one weak binding site per subunit for ATP [Kasprzak, A. and Kochman, M. (1980) Eur. J. Biochem. 104, 443-450]. The reaction of the ATP analog 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine with rabbit aldolase A results in linear inactivation of enzyme with respect to covalent linkage of fluorescent label. The enzyme is completely protected against modification in the presence of saturating covalent binding (k2 = 0.033 min-1) is preceded by a fast reversible binding step (Ki = 6.8 mM). Chemical modification of aldolase leads to formation of stable N epsilon (4-carboxybenzenesulfonyl-lysine (Cbs-Lys) and O-(4-carboxybenzenesulfonyl-tyrosine (Cbs-Tyr) derivatives. Almost all Cbs-Lys was found in the N-terminal CNBr peptide (CN-1), whereas Cbs-Tyr was present both in the N-terminal (CN-1) and C-terminal (CN-2) peptide. From carboxypeptidase digestion and tryptic peptide analysis, Cbs-Lys was localized in position 107, a small part of Cbs-Tyr was detected in position 84, and the majority of Cbs-Tyr was found in the C-terminal position Tyr-363. We conclude that the covalent binding of the ATP analog occurs at the mononucleotide tight-binding site of aldolase and is associated with modification of Lys-107 and Tyr-363. This conclusion is based on the measurements of enzymatic activity loss as a function of ATP analog incorporation as well as on previous data. It is postulated that Lys-107, which is the C-6 phosphate binding site for fructose-1,6-P2, is in close proximity to the functionally important Tyr-363. The rather small extent of modification of Tyr-84 (0.15 mol/subunit), is due either to nonspecific protein modification or labeling of the weak mononucleotide binding site.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D010443:Peptide Chain Termination, Translational; D010455:Peptides / Q000302:isolation & purification; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D010455:Peptides; C029342:5'-(4-fluorosulfonylbenzoyl)-1,N(6)-ethenoadenosine; D005634:Fructose-Bisphosphate Aldolase; D000241:Adenosine