ACTIVE-SITE-DEPENDENT ELIMINATION OF 4-NITROPHENOL FROM 4-NITROPHENYLALYLPHOSPHONYL SERINE-PROTEASE ADDUCTS

Citation
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
Citations number
49
Categorie Soggetti
Chemistry
ISSN journal
00027863
Volume
120
Issue
2
Year of publication
1998
Pages
258 - 263
Database
ISI
SICI code
0002-7863(1998)120:2<258:AEO4F4>2.0.ZU;2-8
Abstract
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.