Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase.
The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D004099:Dihydroxyacetone Phosphate / Q000378:metabolism; D004983:Ethanolamines; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D009132:Muscles / Q000201:enzymology; D009943:Organophosphorus Compounds; D011485:Protein Binding; D011817:Rabbits
D000345:Affinity Labels; D000596:Amino Acids; D004983:Ethanolamines; D009943:Organophosphorus Compounds; D006639:Histidine; C020161:N-bromoacetylethanolamine phosphate; D004099:Dihydroxyacetone Phosphate; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine; D003545:Cysteine