Active-Site-Dependent Elimination of 4-Nitrophenol from 4-Nitrophenyl Alkylphosphonyl Serine Protease Adducts

Chymotrypsin and subtilisin BPN‘ can be inhibited by bis(4-nitrophenyl) methylphosphonate (NMN) and bis(4-nitrophenyl) propylphosphonate (NPN) very efficiently with second-order rate constants, k i / K i, between 544 and 4300 M - 1 s - 1 at 25.0 ± 0.1 °C at the pH maxima. The second-order rate constants for the inhibition of trypsin are 26.3 ± 1.4 M - 1 s - 1 with NMN and 891 ± 14 M - 1 s - 1 with NPN at pH 8.3 and 25.0 ± 0.1 °C. A second stoichiometric equivalent 4-nitrophenol is also lost from 4-nitrophenyl alkylphosphonyl adducts of chymotrypsin but not from trypsin and subtilisin BPN‘. Elimination of 4-nitrophenol from the propylphosphonyl adduct is at a rate only about twice the rate of hydrolysis of a comparable phosphonate diester, whereas 4-nitrophenol is eliminated 270 times faster from the methylphosphonyl adduct of chymotrypsin. The activation enthalpies, in kcal/mol, for 4-nitrophenol elimination from 4-nitrophenyl alkylphosphonylchymotrypsin are 15.0 ± 1.3 for the propyl derivative, 16.4 ± 0.5 for the methyl derivative in H 2 O and 18.0 ± 0.5 in D 2 O. The activation entropies, in cal mol - 1 K - 1, are −29.7 ± 2.4 for the propyl derivative, −14.8 ± 0.5 for the methyl derivative in H 2 O, and −10.3 ± 0.3 for the methyl derivative in D 2 O. Partial solvent isotope effects for the elimination of 4-nitrophenol from 4-nitrophenyl methylphosphonylchymotrypsin give best fits to two-site proton models: These give primary isotope effects between 1.9 and 2.0 (φ 1 ⧧ = 0.52 ± 0.14 or 0.49 ± 0.07) for a proton in flight, possibly from the water attacking at phosphorus to the catalytic His, and an α-secondary effect of 1.3 (φ 2 ⧧ = 0.75 ± 0.20) or a term for solvent contribution of 1.25 (Φ = 0.80 ± 0.10). The secondary β-deuterium isotope effect on the elimination of the second 4-nitrophenol from the adduct of chymotrypsin with NMN-l 3 (l = h or d ) is 0.94 ± 0.2 possibly of hyperconjugative origin. The occurrence and mechanisms of secondary reactions in phosphonylated serine protease enzymes are markedly different from those in phosphonylated cholinesterases.