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Analysis and elimination of protein perturbation in infrared difference spectra of acyl-chymotrypsin ester carbonyl groups by using 13C isotopic substitution.

ABSTRACT: I.r. spectroscopy has been applied to the study of hydrogen-bonding of the unique ester carbonyl group of acylchymotrypsins in the oxyanion hole of the enzyme. This catalytic device provides electrophilic stabilization of negative charge in the transition states and tetrahedral intermediates along the reaction pathway. The use of 13C isotope substitution of the ester carbonyl group reinforces the previous observation [White & Wharton (1990) Biochem. J. 270, 627-637] that the ester carbonyl group is significantly polarized in the ground state by hydrogen bonding in the oxyanion hole. I.r. difference spectra of [carbonyl-12C]-minus [carbonyl-13C]-cinnamoyl-chymotrypsin as well as each of these acylenzymes minus free enzyme are reported. These spectra show that the contribution of protein perturbation (i.e. spectral features that arise from the enzyme which is distorted on acylation) in [carbonyl-12C]cinnamoyl-chymotrypsin minus free enzyme spectra is significant. The contribution of the perturbation components of the spectra is pH-dependent and can represent up to 50% of the total absorbance in the spectral region from 1690 to 1740 cm-1. Use of the isotopic difference method has allowed problems associated with protein perturbation to be eliminated. Similar difference spectra are presented for dihydrocinnamoyl-chymotrypsin. In this case the effect of perturbation is very marked and leads to the cancellation of the band assigned to the non-bonded conformation of the acyl group which has previously only been observed at higher pH. The isotopic difference method again proves reliable and shows that the frequency difference previously used to calculate the ground-state electronic strain induced by the oxyanion-hole catalytic device is not affected by the perturbation, although the amplitudes of the spectral features are different. A study of the deacylation of cinnamoyl-chymotrypsin in water and deuterium oxide using both u.v. and i.r. spectroscopies has confirmed that the use of deuterium oxide as solvent has no serious effect on the deacylation behaviour of the enzyme. I.r. bands assigned to nonproductive and productive conformers decline identically during deacylation, which shows that the conformers are in dynamic exchange on the reaction time-scale.

SUBMITTER: White AJ

PROVIDER: S-EPMC1133161 | biostudies-other | 1992 Oct

REPOSITORIES: biostudies-other

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Project description:The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly (13)C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular (13)C-(13)C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D (1)H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for (13)C T1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ~200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks, which are often difficult to resolve in the parent 2D spectra.

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Analysis and elimination of protein perturbation in infrared difference spectra of acyl-chymotrypsin ester carbonyl groups by using 13C isotopic substitution.

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