Vibrational spectra of scissile bonds in enzyme active sites: a resonance Raman study of dithioacylpapains

Abstract

The resonance Raman (RR) spectra of six transient dithioacylpapains have been obtained. For five of these intermediates the dithioacyl group comprises an N-acylglycine dithioester, RC(=O)NHCH,C(=S)S--, while the sixth is a benzoyloxyacetic acid dithioester, C6H5C(=O)OCH2C-(=S)S--. The R R spectra indicate that, for the great majority of molecules in each dithioacylpapain population, the vibrational properties of the dithioester center undergoing catalytic attack are markedly perturbed. By analogy with the R R spectra of models (the corresponding dithioacyl ethyl esters) and by noting the effects of amide hydrogen-deuterium exchange, the source of the perturbation is identified as an intramolecular interaction between the acyl's amide (for the five N-acylglycine dithioester groups) or ester (for the benzoyloxyacetic acid dithioester group) moiety and the dithioester that forms the dithioacylpapain linkage. The R R and ab-I t was demonstrated recently (Storer et al., 1979) that the resonance Raman (RR) spectrum of a dithioacylpapain enables us to monitor the vibrational spectrum of the bonds undergoing catalytic transformation in the enzyme's active site. During catalysis, a transient -C-C(=S)S-linkage is formed in which the substrate and the enzyme contribute the -C-C-(=S)-and -S-moieties, respectively (Lowe & Williams, 1965). The dithioester group has a A , ,near 315 nm, and by use of laser excitation in the 330-nm region, R R bands can be detected that contain contributions from the stretching motions of the C=S bond, which is undergoing nucleophilic attack in the enzyme's active site, and the C-S bond, which is being cleaved during enzymolysis. The R R spectrum of a dithioacyl-enzyme can be used to form a detailed understanding of the structure of the labile group in the active site. However, spectral interpretation was hampered initially by the lack of background spectroscopic information concerning dithioesters. Thus, we instigated a series of infrared, Raman, and RR spectroscopic studies aimed at providing the material required to interpret the R R spectra of dithioacyl-enzymes. The present paper can only be understood by reference to the spectroscopic work and we summarize the important conclusions from these studies as follows:( 1 ) Simple dithioesters of the type CH3C(=S)SCH3 have an intense H -H* electronic transition near 307 nm, and laser excitation within this transition gives rise to intense RR features near 590 and 1195 cm-l. By means of extensive isotopic substitution and a normal coordinate treatment (Teixeira-Dias et al., 1982), these bands are assigned to modes which, for the 590-cm-' feature, have high contributions from stretching motions of the C-C, C=S, and C-S bonds about the C-C-(=S)-S group and, for the 1195-cm-I feature, have a major contribution from C=S stretching coupled to a smaller contribution from C-C stretching. The vibrational spectral regions sorption spectra of the dithioacyl-enzymes are unchanged between pH 4 and 9, but below pH 4 there are marked spectral alterations which o...

Conformational states of N-acylglycine dithioesters in solution: resonance Raman studies of isotopically substituted models for enzyme-substrate complexes

“…CM-' solutions, to an A conformer, we must now take into account the FTIR evidence that a substantial population of a Cs conformer occurs in CC14 (and other non-H-bonding solvents). This in no way alters the earlier conclusions concerning enzyme-bound substrates (Ozaki et al, 1982a;Huber et al, 1982) since the spectral signatures of the A and C5 conformers are very similar in the 1050-1200-~m-~ region. The 1 174-cm-' features seen in the C C 4 solutions of 1 and 2 (Figures 8 and 6 ) are due to predominately Cs conformers while the corresponding peaks in CH3CN solution at 1165 cm-' are due to predominately A type conformers.…”

“…Discussion of the quantitative nature of geometric strain or distortion has been hampered by the lack of precise experimental evidence on the conformational states of substrates bound in catalytically viable complexes. However, we have shown recently that resonance Raman (RR) spectroscopy can provide such information (Huber et al, 1982;Ozaki et al, 1982a). In the present paper N-acylglycine ethyl dithioesters in solution are taken as standards for relaxed, unperturbed conformers with which to compare the corresponding N-acylglycine dithioacylpapains.…”

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives

“…The thionoester substrates were prepared as described by Ozaki et al (1982) and by Carey et al (1984).…”

Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters

“…The thiono ester substrates [N-benzoylglycine methyl thiono ester and N-(P-phenylpropiony1)glycine methyl thiono ester] were synthesized as described by Ozaki et al (1982). The thiol acids were synthesized as described by Cronyn & Jiu (1952), and the oxygen acids and esters were prepared from the acid troduced for the latter substrate is thought to be due to a slow reaction involving the hydrogen sulfide produced as a primary product in the reaction (see below).…”

“…The sulfur-for-oxygen substitution in ester substrates results in the formation of dithioacyl enzymes that are chromophoric ( A, , , near 315 nm) (Lowe & Williams, 1965a) and that are ideally suited for the application of resonance Raman spectroscopy (Storer et al, 1979). The spectroscopic data demonstrate that all glycine-based thiono ester substrates tested with a wide variety of cysteine proteinases form a single homogeneous conformational population of active-site-bound dithioacyl groups, designated conformer B, in which the glycine's amide N atom comes into close contact with the thiol S atom of cysteine-25 (Ozaki et al, 1982; Huber et al, 1982;Storer et al, 1983;Carey et al, , 1984b Brocklehurst et al, 1984;Varughese et al, 1984). There is evidence from some of these studies that the strength of the N .…”