Inhibition of rabbit muscle aldolase by phosphorylated aromatic compounds.
The interactions of the phosphorylated derivatives of hydroquinone (HQN-P2), resorcinol (RSN-P2), 4-hydroxybenzaldehyde (HBA-P) and 2, 4-dihydroxybenzaldehyde (DHBA-P; phosphate group at position 4) with fructose bisphosphate aldolase were analysed by enzyme kinetics, UV/visible difference spectroscopy and site-directed mutagenesis. Enzyme activity was competitively inhibited in the presence of HQN-P2, RSN-P2 and HBA-P, whereas DHBA-P exhibited slow-binding inhibition. Inhibition by DHBA-P involved active-site Schiff-base formation and required a phenol group ortho to the aldehyde moiety. Rates of enzyme inactivation and of Schiff-base formation by DHBA-P were identical, and corresponded to 3.2-3.5 DHBA-P molecules covalently bound per aldolase tetramer at maximal inactivation. Site-directed mutagenesis of the active-site lysine residues at positions 107, 146 and 229 was found to be consistent with Schiff-base formation between DHBA-P and Lys-146, and this was promoted by Lys-229. Mutation of Glu-187, located vicinally between Lys-146 and Lys-229 in the active site, perturbed the rate of Schiff-base formation, suggesting a functional role for Glu-187 in Schiff-base formation and stabilization. The decreased cleavage activity of the active-site mutants towards fructose 1, 6-bisphosphate is consistent with a proton-transfer mechanism involving Lys-229, Glu-187 and Lys-146.
Blonski
C
C
Groupe de Chimie Organique Biologique, UMR 5623, Bât IIR1, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France.
De Moissac
D
D
Périé
J
J
Sygusch
J
J
D000614:Aminocaproates / Q000378:metabolism; D000818:Animals; D001547:Benzaldehydes / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors*; D006873:Hydroquinones / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010755:Organophosphates / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D012118:Resorcinols / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
C105855:2,4-dihydroxybenzaldehyde 4-phosphate; C105857:4-hydroxybenzaldehyde phosphate; D000614:Aminocaproates; D001547:Benzaldehydes; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D010755:Organophosphates; D011994:Recombinant Proteins; D012118:Resorcinols; C105856:hydroquinone 1,4-bisphosphate; C105858:resorcinol bisphosphate; D005634:Fructose-Bisphosphate Aldolase; D008239:Lysine