Permeable membrane/mass spectrometric measurement of solvent proton/deuterium, carbon-12/carbon-13, and oxygen-16/oxygen-18 kinetic isotope effects associated with .alpha.-chymotrypsin deacylation: evidence for reaction mechanism plasticity

Abstract

We have measured, by permeable membrane/mass spectrometry, the 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects (kie) associated with acyl-alpha-chymotrypsin hydrolysis and transesterification. The hydrolysis of alpha-chymotrypsinyl 2-furoate has a 12C/13C kie of approximately 1.06. Transesterification of the same acyl enzyme shows 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects of 1.015 (0.003), 1.01-1.02, and 2.226 (0.007), respectively. From the temperature independence of the 16O/18O transesterification kinetic isotope effect and kinetic data reported elsewhere [Wang, C.-L. A., Calvo, K. C., & Klapper, M. H. (1981) Biochemistry 20, 1401-1408], we conclude that there are two active forms of acylchymotrypsin. We also propose that formation of the tetrahedral intermediate is the rate-limiting step in both hydrolysis and transesterification and that the position of the transition state in the transesterification is closer to the starting enzyme ester while that for the hydrolytic reaction is closer to the tetrahedral intermediate. These results are discussed in terms of reaction mechanism plasticity.

Measuring Kinetic Isotope Effects in Enzyme Reactions Using Time-Resolved Electrospray Mass Spectrometry

“…To the best of our knowledge, this represents the first competitive heavy-atom KIE measurement in the presteady state. While there are no directly comparable measurements in the literature, this value is within the range determined for deacylation of chymotrypsin using a single-turnover approach (≤1.08, depending on ethanol concentration) . The value is significantly higher than the 1.030 ± 0.002 steady state 13 ( V / K ) value reported by Hess and co-workers; however, that measurement would have been a convolution of the primary KIEs for acylation and deacylation and would also have been influenced by any change in the ratio of the acylation/deacylation rates resulting from isotopic substitution.…”

“…While there are no directly comparable measurements in the literature, this value is within the range determined for deacylation of chymotrypsin using a single-turnover approach (≤1.08, depending on ethanol concentration). 41 The value is significantly higher than the 1.030 ± 0.002 steady state 13 (V/K) value reported by Hess and co-workers; 40 however, that measurement would have been a convolution of the primary KIEs for acylation and deacylation and would also have been influenced by any change in the ratio of the acylation/deacylation rates resulting from isotopic substitution. The magnitude of our acylation-specific KIE measurement indicates a late transition state for loss of paranitrophenolate from the tetrahedral acyl-enzyme complex, consistent with strong stabilization of the oxyanion.…”

Catalytic versatility of Bacillus pumilus .beta.-xylosidase: glycosyl transfer and hydrolysis promoted with .alpha.- and .beta.-D-xylosyl fluoride

“…Glycosylases appear not to be alone in this respect. Evidence has been reported for the plasticity of the reaction mechanism involved in the deacylation of acyl-a-chymotrypsin, with the transition state occurring earlier in transesterification than in hydrolysis (Mishra & Klapper, 1986), in the hydrolytic cleavage of minimal vs. larger aryl groups from a-chymotrypsin (Stein et al, 1983), and in reactions catalyzed with different substrates by certain dehydrogenases (Scharschmidt et al, 1984;Hermes et al, 1984). However, the evidence appears particularly striking in the case of glycosylases where stereochemically different reactions have by now been found to be catalyzed by a considerable number of enzymes.…”

Preferential Alternatives to Returning All Crop Residues as Biochar to the Crop Field? A Three-Source 13C and 14C Partitioning Study

“…Dissolved organic C in biochar extracts was determined using an elemental analyzer (multi EA 4000 for Macro-Elemental Analysis, Analytik Jena, Germany), and the total contents of C and N in the solid biochar were assessed following the LECO Combustion Analysis methods . Biochar δ 13 C value was determined using a stable isotope ratio mass spectrometer (253 Ultra, Thermo Scientific) with ±0.30‰ accuracy and was calculated from where R s and R V‑PDB represent the biochar ratio of 13 C/ 12 C and the Vinna Pee Dee Belemnite (V-PDB) standard, respectively.…”