title: Resonance Raman spectroscopic and kinetic consequences of a nitrogen ... sulphur enzyme-substrate contact in a series of dithioacylpapains.
abstract: The resonance Raman (RR) spectroscopic, conformational, and kinetic properties of six dithioacylpapain intermediates have been examined. Five of the intermediates are of the form N-(methyloxycarbonyl)-X-glycine-C(= S)S-papain, where X is L-phenyl-alanine, D-phenylalanine, glycine, L-phenylglycine, or D-phenylglycine. The sixth intermediate is N-phenylacetyl-glycine-C(= S)S-papain. Throughout the series there is an approximately 50-fold variation in kcat, the rate constant for deacylation, and a 1750-fold variation in kcat/KM. Existing RR spectra structure correlations allow us to define the torsional angles in the NH-CH2-C(= S)-S-CH2-CH fragment of the functioning intermediates. The values of these angles for each bound substrate appear to be very similar, with the substrates assuming a B-type conformer such that the nitrogen atom of the P1 glycine residue is cis to the thiol sulphur atom of cysteine-25. For each intermediate, the C(= S)S-CH2CH torsional angle is approximately -90 degrees, whereas for the SCH2-CH torisonal angle the cysteine-25 thiol sulphur (S) and cysteine-25 C alpha hydrogen (H) atoms are approximately trans. The three acyl-enzymes with the lowest catalytic rate constants, viz. N-(methyloxycarbonyl)-glycine-glycine-, N-(methyloxycarbonyl)-L-phenylglycine-glycine-, or N-(phenylacetyl)-glycine-dithioacylpapains, have atypical RR spectra in that they show a feature of medium intensity in the 1,085-cm-1 region. This band is sensitive to NH to ND exchange of the P1 glycine residues' (-NH-) function and, thus, the corresponding mode involves an excursion of the NH hydrogen. It is hypothesized that the high intensity is due to a particularly strong interaction between the P1 glycine nitrogen atom and the thiol sulphur of cysteine-25, which also has the effect of retarding deacylation, because the nitrogen . . . sulphur contact has to be broken in the rate-determining step.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
authors.forename: P J
authors.initials: PJ
authors.lastname: Tonge
authors.forename: B
authors.initials: B
authors.lastname: Gour-Salin
authors.forename: P
authors.initials: P
authors.lastname: Lachance
authors.forename: A C
authors.initials: AC
authors.lastname: Storer
authors.forename: P R
authors.initials: PR
authors.lastname: Carey
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010206:Papain / Q000737:chemistry*; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D010206:Papain