Steady-state carbon-13 nuclear magnetic resonance spectra of acyl .alpha.-chymotrypsin

Abstract

When [13C]carbonyl-enriched p-nitrophenyl 5-n-propyl-2-furoate is incubated with alpha-chymotrypsin, a new peak appears in the 13C NMR spectrum. On the basis of its position and the fact that it is "chased" with unlabeled substrate, we conclude that this new signal is due to the acyl-enzyme intermediate. In spectra taken during steady-state turnover, the acyl-enzyme ester carbonyl 13C chemical shift displays a pH dependence that fits to a titration curve with an apparent pK of 7.1 (0.1). The apparent pK of the kcat vs. pH curve for enzyme-catalyzed hydrolysis of the same substrate under conditions differing only in reactant concentration is 7.0 (0.1). We have found no spectral evidence for a tetrahedral intermediate.

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

“…The results we have presented here and in previous papers (McWhirter et al, 1985;Wang et al, 1981) permit us a more detailed, albeit still qualitative, picture of the a-chymotrypsin deacylation reaction mechanism. The temperature independence of the 160/180 kie together with our earlier analysis of the nonlinear Arrhenius plots observed by us and others (Baggott & Klapper, 1976;Wang et al, 1981 Laidler, 1967; Click, 1971; Wedler et al, 1975) supports the proposal of at least two active acyl enzyme forms in a temperature-dependent equilibrium.…”

“…The ethanol transesterification of -chymotrypsinyl 2-furoate has a temperature-independent 160/lsO kie of 1.015 (0.003), from which we conclude that the observed Arrhenius nonlinearity of the transesterification, if not hydrolysis as well, is consistent with a temperature-dependent equilibrium between two acyl enzyme forms that breakdown at different rates. There has been spectroscopic evidence for two forms of the acyl enzyme (MacClement et al, 1981;Shah et al, 1984;McWhirter et al, 1985), but it is not known whether the spectrally and kinetically observed heterogeneities are related.…”

“…After distillation of the excess thionyl chloride, we purified the furoyl chloride by vacuum distillation (bp -110 °C at 30 mmHg). The [13C]carbonyl-enriched NF was synthesized as described elsewhere (McWhirter, et al, 1985) and the lsO-enriched ethanol by the method of Sawyer (1972). MA-Dimethylformamide was treated with P2Os for 3 days, with KOH for 3 additional days, and then distilled under vacuum.…”

Time-resolved X-ray diffraction studies of enzymes under cryoconditions

“…Then, enzyme intermediates might exist in more than one structural form, or con formational isomers may possibly interconvert. Some experimental evidence has been provided by 13C n m r studies of acyl-chymotrypsin (McClement et al 1981;McWhirter et al 1985).…”