ASP70 IN THE PERIPHERAL ANIONIC SITE OF HUMAN BUTYRYLCHOLINESTERASE

The goal of this work was to determine what amino acids at the mouth o f the active-site gorge are important for the function of human butyry lcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G2 83D were expressed in human embryonal kidney cells and the secreted en zymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 w as mutated to G, the same mutation as in the naturally occurring atypi cal butyrylcholinesterase, the affinity for positively charged substra tes and positively charged inhibitors decreased 5-30-fold. The D70G mu tant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine . Thus, though k(cat) was the same for wild-type and D70G mutant, bein g 24000 min(-1) at low butyrylthiocholine concentrations (0.01-0.1 mM) , it failed to increase for the D70G mutant at 40 mM butyrylthiocholin e, whereas it increased threefold for wild type. The D70G mutant was m ore sensitive to changes in salt concentration, its catalytic rate dec reasing more than that of the wild type. The D70G mutant appeared to h ave a greater surface negative charge than wild type suggesting that t he D70G mutant had a conformation different from that of the wild type . That D70 affects the function of butyrylcholinesterase, together wit h its location at the mouth of the active-site gorge, supports the hyp othesis that D70 is a component of the peripheral anionic site of buty rylcholinesterase. Mutants containing aromatic amino acids at the mout h of the gorge had increased binding affinity for propidium and fascic ulin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyr ylcholinesterase.