ROLE OF ASPARTATE-70 AND TRYPTOPHAN-82 IN BINDING OF SUCCINYLDITHIOCHOLINE TO HUMAN BUTYRYLCHOLINESTERASE

The atypical Variant of human butyrylcholinesterase has Gly in place o f Asp 70, Patients with this D70G mutation respond abnormally to the m uscle relaxant succinyldicholine, experiencing hours of apnea rather t han the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the Liter ature is why the atypical variant has a 10-fold increase in K-m for co mpounds with a single positive charge but a 100-fold increase in K-m f or compounds with two positive charges. We mutated residues Asp 70, Tr p 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in ma mmalian cells. Steady-state kinetic parameters far hydrolysis of butyr lthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had ident ical k(cat) values for all substrates. Molecular modeling and molecula r dynamics suggested that succinyldicholine could bind in two consecut ive orientations in the active site gorge, formation of one complex ca used a conformational change in the omega loop involving Asp 70 and Tr p 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this con tention, Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus exp laining the 10- and 100-fold increases in K-m in the D70G mutant.