Protonic reorganization in catalysis by serine proteases: acylation by small substrates

Abstract

Synthesis of N-Ac-Ala-Gly-Gly-OMe. To 775 mg (3.8 mmol) of Lalanylglycylglycine (Ala-Gly-Gly, Nutritional Biochemicals Corp., Cleveland, Ohio) slurried in 125 mL of dry methanol at -60 °C was added 10.0 mL (139 mmol) of thionyl chloride in 50 mL of CHC13. The solution was allowed to warm to room temperature with constant stirring, after which the solvents were removed in vacuo, followed by redissolution of the residue in 50 mL each of pyridine and water. At 0 °C, 4.0 mL (116 mmol) of acetic anhydride was added with vigorous stirring, followed by a second 4.0 mL 15 min later. The reaction proceeded 30 min longer, followed by evaporation and chromatography on silica gel (14.5 X 3.8 cm) in 300-mL steps of 5, 10, and 15% methanol-chloroform. The acetylated tripeptide methyl ester, A'-Ac-Ala-Gly-Gly-OMe, eluted in the last two steps: yield 530 mg (54%); mp 133-135 °C; MS parent peak at 259 (A + \ j A = 0.124 ± 0.004; caled., 0.124), fragmentation peaks at m/e 171, 144, 114, and 87 for CH3CONHCHCH3CONHCH2CO+, +NCH2C0NHCH2C02CH3, CH3CONHCHCH3CO+, and +NCH2C02CH3, respectively; 'H NMR (2H20, DSS internal reference) 4.28 (quartet, C"-H of Ala), 4.04 and 3.96 (2s, a-CH2 of each Gly), 3.75 (s, methyl ester CH3), 2.03 (s, CH3 of acetyl group), 1.39 ppm (doublet /3-CH3 of Ala).Procedures. Enzyme alkylation, assay, and purification have been described in the preceding paper.3 p-Nitrophenyl acetate kinetics were measured spectrophotometrically on a Unicam SP 1800 instrument at pH 7.16 (0.09 M phosphate), 25 °C, 6.9% CH3CN (to dissolve the substrate), at 347.8 nm where an isosbestic point for nitrophenol was used: At = 5.06 X 103 M"1 cm'1. The pH-rate profile was measured in phosphate buffers as well.Ac-Tyr-OEt and A'-Ac-Ala-Gly-Gly-OMe kinetics were measured with a Radiometer pH-stat system at pH 8.0, 0.1 M, KC1, 25 °C. All kinetic data were processed on a Data General Nova 1220 minicomputer by either linear least squares or a nonlinear regression analysis written by . H. Klapper based on curfit by Bevington.10

Catalysis by human leukocyte elastase: the proton inventory as a mechanistic probe

“…Clearly, the experimental data cannot be described by this model. Furthermore, the data were fit by a polynomial regression method to determine the statistical significance, by an F test, of each additional polynomial term, or proton bridge [Schowen and Schowen (1982), especially pp 559-567; Venkatasubban & Schowen, 19851. For the proton inventory of MeOSuc-Ala-Ala-Pro-Val-HLE deacylation, the linear and quadratic terms are significant at the 99.9% confidence level, while cubic and higher terms are significant at less than an 80% confidence level.…”

“…An expression for the proton inventory for these reactions can be derived by starting from eq 6 and two mechanistic features of serine protease catalyzed reactions (Stein,198%): (i) dissociation constants of complexes formed from serine proteases and substrates or inhibitors are frequently lower in magnitude in D20 than in H 2 0 (Le., DKm DKi > l), and (ii) the transition state corresponding to k2/Ks may be virtual. This expression was derived from eq 6 in earlier studies (Stein, 1985c,d) and is 1 : : : Z1 [Schowen and Schowen (1982), especially pp 559-5671 is a composite, transition-state fractionation factor that reflects the generalized solvent reorganization that occurs during substrate binding (Kresge & Schowen, 1975). Z will be greater than 1 and similar in magnitude to dissociation constant solvent isotope effects.…”

“…x e proton inventory technique is an extension of the more routinely used solvent isotope effect; the latter is determined by measuring reaction rates in H 2 0 and D 2 0 and expressed as a ratio of rate constants, typically kH/kD and abbreviated here as Dk, while the former is determined by measuring reaction rates in mixtures of the isotopic waters and expressed graphically as the dependence of rate on the mole fraction of solvent deuterium n. The size of the solvent isotope effect and shape of the proton inventory are diagnostic of reaction mechanism [Schowen and Schowen (1982), especially pp 559-567; Venkatasubban & Schowen, 19851 and, upon detailed analysis, often lead to novel mechanistic insights (Harmony et al, 1975;Hunkapiller et al, 1976;Pollock et al, 1973;Matta & Toenjes, 1985;Stein, 198%).…”

Natural sialoside analogues for the determination of enzymatic rate constants

“…1 In addition with respect to the corresponding enzymatic systems, it has been reported that p-nitrophenyl acetate initially acylates a-lytic protease on its active site histidine residue, and that this acylated imidazole species undergoes a subsequent N-O acyl transfer to give the expected acyl-enzyme intermediate. 2 In the case of sialidases, two separate papers appeared in 2003 in which it was proposed that the exo-sialidase family of glycosidase and trans-glycosidases react via a double displacement mechanism and that a tyrosine residue acts as a nucleophile during the enzy-Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6. E-mail: bennet@sfu.ca matic reaction to give a transient sialosyl-enzyme intermediate.…”

Introduction

“…The solved structure of chymotrypsin suggested that a grouping of active site amino acids, Ser195, His, 57, and Asp102, now known as the " charge relay system, " plays a critical role in catalysis by this enzyme. The actual operation of the charge relay system has been demonstrated not only for chymotrypsin but also for a number of other serine proteases (Elrod et al 1976(Elrod et al , 1980Quinn et al 1980 ;Stein et al 1987 ). In the years since these structures appeared, hundreds more have been solved for enzymes of all mechanistic classes.…”