ENANTIOSELECTIVE AND REVERSIBLE INHIBITION OF TRYPSIN AND ALPHA-CHYMOTRYPSIN BY PHOSPHONATE ESTERS

Trypsin is inactivated by the levorotatory enantiomers (most likely P- S) of 4-nitrophenyl 4-H-, 4-CH3-, 4-OCH3-, and 4-Cl-phenacyl methylpho sphonates (PMNs) with second-order rate constants between 231 and 884 M(-1) s(-1). 4-NO2-PMN hydrolyzes before inhibiting the enzyme. The se cond-order rate constants for the inactivation of a-chymotrypsin by th e levorotatory enantiomers of the five PMNs are between 37 000 and 770 000 M(-1) s(-1), and those for the dextrorotatory enantiomers are bet ween 400 and 640 M(-1) s(-1); the enantioselectivity is 90-1880. Speci fic rotation [alpha](22)(D) of the faster-reacting enantiomer of 4-CH3 -PMN with trypsin and ol-chymotrypsin is -30 +/- 6 degrees. P-31 NMR o f the adducts shows a signal at 41.0 ppm, 10 ppm downfield from the pa rent compound. Results of molecular mechanics and dynamics calculation s show that the principal interactions are between the phosphonyl grou p and constituents of the oxyanion hole and between the aromatic fragm ent and residues in the binding regions of the enzymes. Trypsin activi ty returns from its phenacyl methylphosphonyl adducts on the hour time scale and in reversed order to the rates of inactivation within the s eries. Recovery of alpha-chymotrypsin activity from the adducts formed with the (-) enantiomers is on a slower time scale still, whereas its recovery from the adducts formed with the (+) enantiomers is on the s econd to minute time scale. The data support a mechanism of reactivati on involving rate-determining intramolecular displacement of Ser by th e carbonyl hydrate of the phenacyl moiety. The pH-rate profiles for tr ypsin reactivation from its adducts indicate involvement of an ionizab le group with pK(a) similar to 8.0. The pH dependence and solvent isot ope effects are small in most cases. The compounds demonstrate favorab le properties for controllable and temporary modulation of enzyme acti vity.