Characterization of trans- and cis-5-Methylthienylacryloyl Chymotrypsin Using Raman Difference Spectroscopy, NMR, and Kinetics: Carbonyl Environment and Reactivity

P. J. Tonge, P. R. Carey, R. Callender, H. Deng, I. Ekiel, D. R. Muhandiram

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

The interpretation of the resonance Raman (RR) data for chromophoric acyl serine proteases is rendered complex due to the fact that the ≈350-nm laser beam used to generate the RR spectrum causes the substrate to photoisomerize in the active site. As a result, a steady-state population of dark (present before irradiation) and light (photoinduced) conformera is set up. In the present work, 2D NMR experiments are used to confirm that the photoinduced conformer of 5-methylthienylacryloyl Chymotrypsin is cis about the acryloyl —C=C— linkage, whereas it is trans for the dark conformer. Using 488.0- or 514.5-nm excited Raman difference spectroscopy the normal Raman spectra, under nonphotolyzing conditions, of the pure cis and trans forms of the acyl Chymotrypsins are reported for the first time. A number of characteristic bands for the two forms are identified, e.g., the bands at 1616, 1470, and 1534 cm−1 for the trans form of the intermediate occur at 1611, 1419, and 1527 cm−1 in the cis form. In particular vc=o for the acyl carbonyl occurs at 1727 and 1697 cm−1 for the cis and trans forms, respectively. The two forms have markedly different reactivities with the trans isomer deacylating 5000 times faster than the cis. These spectroscopic and kinetic properties contrast sharply with the behavior of the model compound 5-methylthienylacryloyl methyl ester. The cis and trans forms of this ester in CCl4 have values for vc=o at 1720 ± 2 cm−1, and the intrinsic reactivities of the cis and trans methyl esters to base catalyzed hydrolysis are very similar, with kOH- of 0.0062 and 0.013 s−1 in 1.0 M NaOH, respectively. Thus, the differential reactivities and vc=o observed for the cis and trans acyl Chymotrypsins are caused by differential interactions with the enzyme's active site and are not due to the intrinsic properties of the cis and trans form of the acyl group.

Original languageEnglish (US)
Pages (from-to)8757-8762
Number of pages6
JournalJournal of the American Chemical Society
Volume115
Issue number19
DOIs
StatePublished - Sep 1 1993

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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