Im. Kovach et Ej. Enyedy, ACTIVE-SITE-DEPENDENT ELIMINATION OF 4-NITROPHENOL FROM 4-NITROPHENYLALYLPHOSPHONYL SERINE-PROTEASE ADDUCTS, Journal of the American Chemical Society, 120(2), 1998, pp. 258-263
Chymotrypsin and subtilisin BPN' can be inhibited by bis(4-nitrophenyl
) methylphosphonate (NMN) and bis(4-nitrophenyl) propylphosphonate (NP
N) very efficiently with second-order rate constants, k(i)/K-i, betwee
n 544 and 4300 M-1 s(-1) at 25.0 +/- 0.1 degrees C at the pH maxima. T
he 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 degrees C. A second stoichiometric equivalent 4-nitr
ophenol is also lost from 4-nitrophenyl alkylphosphonyl adducts of chy
motrypsin but not from trypsin and subtilisin BPN'. Elimination of 4-n
itrophenol from the propylphosphonyl adduct is at a rate only about tw
ice the rate of hydrolysis of a comparable phosphonate diester, wherea
s 4-nitrophenol is eliminated 270 times faster from the methylphosphon
yl adduct of chymotrypsin. The activation enthalpies, in kcal/mol, for
4-nitrophenol elimination from 4-nitrophenyl alkylphosphonylchymotryp
sin are 15.0 +/- 1.3 for the propyl derivative, 16.4 +/- 0.5 for the m
ethyl derivative in H2O and 18.0 +/- 0.5 in D2O. The activation entrop
ies, in cal mol(-1) K-1, are -29.7 +/- 2.4 for the propyl derivative,
-14.8 +/- 0.5 for the methyl derivative in H2O, and -10.3 +/- 0.3 for
the methyl derivative in D2O. Partial solvent isotope effects for the
elimination of 4-nitrophenol from 4-nitrophenyl methylphosphonylchymot
rypsin give beat fits to two-site proton models: These give primary is
otope effects between 1.9 and 2.0 (phi(1) double dagger = 0.52 +/- 0.1
4 or 0.49 +/- 0.07) for a proton in flight, possibly from the water at
tacking at phosphorus to the catalytic His, and an alpha-secondary eff
ect of 1.3 (phi(2) double dagger = 0.75 +/- 0.20) or a term for solven
t contribution of 1.25 (Phi = 0.80 +/- 0.10). The secondary beta-deute
rium isotope effect on the elimination of the second 4-nitrophenol fro
m the adduct of chymotrypsin with NMN-1(3) (1 = h or d) is 0.94 +/- 0.
2 possibly of hyperconjugative origin. The occurrence and mechanisms o
f secondary reactions in phosphonylated serine protease enzymes are ma
rkedly different from those in phosphonylated cholinesterases.