Butyrylcholinesterase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-related neutral esters

Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholinesterase (BuChE). Catalytic parameters were determined in 100 mM Tris buffer, pH 7.4, in the presence and absence of metal cations. The presence of Ca²⁺ or Mg²⁺ (<100 mM) in buffer did not change the K(m), but accelerated the rate of hydrolysis of aspirin by wild-type or D70G mutant BuChE by 5-fold. Turnover numbers were of the order of 5000-12 000 min^-1 for the wild-type enzyme and the D70G and D70K enzymes in 100 mM Tris, pH 7.4, containing 50 mM CaCl₂ at 25°C; K(m) values were 6 mM for wild-type, 16 mM for D70G and 38 mM for D70K. People with 'atypical' BuChE have the D70G mutation. The apparent inhibition seen at high aspirin concentration was not due to inhibition by excess substrate but to spontaneous hydrolysis of aspirin, causing inhibition by salicylate. The wild-type and D70G enzymes were competitively inhibited by salicylic acid; the D70K enzyme showed a complex parabolic inhibition, suggesting multiple binding. The effect of salicylate was substrate-dependent, the D70K mutant being activated by salicylate with butyrylthiocholine as substrate. K(m) value for wild-type enzyme was lower than for D70 mutants, suggesting that residue 70 located at the rim of the active site gorge was not the major site for the initial encounter aspirin-BuChE complex. On the other hand, the virtual absence of affinity of the W82A mutant for aspirin indicated that W82 was the major residue involved in formation of the Michaelis complex. Molecular modelling of aspirin binding to BuChE indicated perpendicular interactions between the aromatic rings of W82 and aspirin. Kinetic study of BuChE-catalysed hydrolysis of different acetyl esters showed that the rate limiting step was acetylation. The bimolecular rate constants for hydrolysis of aspirin by wild-type, D70G and D70K enzymes were found to be close to 1x10⁶ M^-1 min^-1. These results support the contention that the electrostatic steering due to the negative electrostatic field of the enzyme plays a role in substrate binding, but plays no role in the catalytic steps, i.e. in the enzyme acetylation. Copyright (C) 1998 Elsevier Science B.V.