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.
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- Angus R. H., Carey P. R., Lee H., Storer A. C. Conformational activation of acylpapains and acylcathepsin B's: resonance Raman and kinetic evidence. Biochemistry. 1986 Jun 3;25(11):3304–3310. doi: 10.1021/bi00359a033. [DOI] [PubMed] [Google Scholar]
- Carey P. R., Angus R. H., Lee H. H., Storer A. C. Identity of acyl group conformations in the active sites of papain and cathepsin B by resonance Raman spectroscopy. J Biol Chem. 1984 Dec 10;259(23):14357–14360. [PubMed] [Google Scholar]
- Huber C. P., Ozaki Y., Pliura D. H., Storer A. C., Carey P. R. Precise structural information for transient enzyme-substrate complexes by a combined X-ray crystallographic-resonance Raman spectroscopic approach. Biochemistry. 1982 Jun 22;21(13):3109–3115. doi: 10.1021/bi00256a012. [DOI] [PubMed] [Google Scholar]
- Lee H., Storer A. C., Carey P. R. Conformational states of N-acylglycine dithioesters in solution: resonance Raman studies of isotopically substituted models for enzyme-substrate complexes. Biochemistry. 1983 Sep 27;22(20):4781–4789. doi: 10.1021/bi00289a026. [DOI] [PubMed] [Google Scholar]
- Ozaki Y., Pliura D. H., Carey P. R., Storer A. C. Vibrational spectra of scissile bonds in enzyme active sites: a resonance Raman study of dithioacylpapains. Biochemistry. 1982 Jun 22;21(13):3102–3108. doi: 10.1021/bi00256a011. [DOI] [PubMed] [Google Scholar]
- Ozaki Y., Pliura D. H., Carey P. R., Storer A. C. Vibrational spectra of scissile bonds in enzyme active sites: a resonance Raman study of dithioacylpapains. Biochemistry. 1982 Jun 22;21(13):3102–3108. doi: 10.1021/bi00256a011. [DOI] [PubMed] [Google Scholar]
- Storer A. C., Angus R. H., Carey P. R. Comparison of the kinetics of the papain-catalyzed hydrolysis of glycine- and alanine-based esters and thiono esters. Biochemistry. 1988 Jan 12;27(1):264–268. doi: 10.1021/bi00401a040. [DOI] [PubMed] [Google Scholar]
- Storer A. C., Carey P. R. Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters. Biochemistry. 1985 Nov 19;24(24):6808–6818. doi: 10.1021/bi00345a012. [DOI] [PubMed] [Google Scholar]
- Storer A. C., Lee H., Carey P. R. Relaxed and perturbed substrate conformations in enzyme active sites: evidence from multichannel resonance raman spectra. Biochemistry. 1983 Sep 27;22(20):4789–4796. doi: 10.1021/bi00289a027. [DOI] [PubMed] [Google Scholar]