Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates

Human butyrylcholinesterase displays substrate activation with positively c harged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site ( PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and act ivation control, the single mutants D70G/Y and Y332F/A/D and the double mut ants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, ETC and succinyldithiocholine, and the neutral ester o -nitrophenyl butyrate was measured. In addition, inhibition of wild-type an d mutant enzymes by tetramethylammonium was investigated, at low concentrat ions of ETC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for acti vation control. The effects of double mutations on D70 and Y332 were comple x. Double-mutant cycle analysis provided evidence for interaction between t hese residues. The category of interaction (either synergistic, additive, p artially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity r eflects both the crosstalk between residues involved in the sequential form ation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS ind uce a conformational change in the active site gorge of butyrylcholinestera se that can alter both substrate binding and enzyme acylation. (C) 1999 Els evier Science B.V. All rights reserved.