α-Helix Dipoles and Catalysis: Absorption and Raman Spectroscopic Studies of Acyl Cysteine Proteases
- 1 January 1996
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 35 (38) , 12495-12502
- https://doi.org/10.1021/bi960649+
Abstract
Raman and absorption spectroscopic data are combined with the deacylation rate constants for a series of acyl cysteine proteases to provide insight into the role of alpha-helix dipoles in rate acceleration. The Raman spectra, obtained by Raman difference spectroscopy, of (5-methylthienyl)acryloyl adducts with papain, cathepsins B and L, and two oxyanion hole mutants of cathepsin B (Q23S and Q23A) show that extensive polarization throughout the pai-electron chain occurs for the bound acyl group in the active sites. A similar result is obtained using the specific chromophoric substrate ethyl 2-[(N-acetyl-L-phenylalanyl)amino]-3-(5-methylthienyl)acrylate. By using 13C = O substitution it is possible to detect the acyl C = O stretching frequency, vC = O, for each acyl enzyme. A correlation between vC = O and log k3, where k3 is the deacylation rate constant, is found where vC = O increases with increasing reactivity. This is exactly the opposite sense to the relationship found for a series of acyl serine proteases [Carey & Tonge (1995) Acc. Chem. Res. 28, 8]. The opposite trend in the direction of the correlation for the acyl cysteine proteases is ascribed to the strong electron polarizing forces in the active site, due principally to the active-site alpha-helix dipole, giving rise to canonical (valence bond) forms of the acyl group which change the hybridization about the C = O carbon atom. A correlation is also observed between the absorption maximum, lambda max, and log k3 for each acyl cysteine protease. As the deacylation rate increases, 214-fold across the series, lambda max red-shifts from 367 to 384 nm. It is proposed that differential interactions between the active site's alpha-helix dipole and the acyl chromophore give rise to the observed changes in lambda max, with the red shift being caused principally by interactions with the excited electronic state, which has a high degree of charge separation, and the dipole. Similar interactions between the dipole and the negatively charged tetrahedral intermediate, which resembles the transition state, are proposed as the source of differential rates in deacylation. It is interesting to note that similar energies are operating in both cases. A shift in lambda max from 367 to 384 nm corresponds to a change in electronic absorption transition energies of 3.2 kcal/mol and a change of deacylation rate constants of 214-fold also corresponds to a change of activation energies of 3.2 kcal/mol.Keywords
This publication has 35 references indexed in Scilit:
- Synthesis of chromophoric dipeptides as substrates for papainBioorganic & Medicinal Chemistry Letters, 1995
- Nonresonance Raman Difference Spectroscopy: A General Probe of Protein Structure, Ligand Binding, Enzymatic Catalysis, and the Structures of Other BiomacromoleculesAnnual Review of Biophysics, 1994
- Observation of a carbonyl feature for riboflavin bound to riboflavin-binding protein in the red-excited raman spectrumJournal of the American Chemical Society, 1993
- Resonance Raman and Fourier transform infrared spectroscopic studies of the acyl carbonyl group in [3-(5-methyl-2-thienyl)acryloyl]chymotrypsin: evidence for artifacts in the spectra obtained by both techniquesBiochemistry, 1991
- Direct observation of the titration of substrate carbonyl groups in the active site of .alpha.-chymotrypsin by resonance Raman spectroscopyBiochemistry, 1989
- The active site of papainJournal of Molecular Biology, 1989
- Structure of papain refined at 1.65 Å resolutionJournal of Molecular Biology, 1984
- Evidence for two acyl group conformations in some furylacryloyl- and thienylacryloylchymotrypsins: resonance Raman studies of enzyme-substrate intermediates at pH 3.0Biochemistry, 1981
- Resonance Raman evidence for substrate reorganization in the active site of papainBiochemistry, 1976
- Structure of crystalline α-chymotrypsinJournal of Molecular Biology, 1970