ACTIVE-SITE PROPERTIES OF THE 3C PROTEINASE FROM HEPATITIS-A VIRUS (AHYBRID CYSTEINE SERINE PROTEASE) PROBED BY RAMAN-SPECTROSCOPY/

Although the HAV 3C proteinase is a cysteine protease, it displays an active site configuration which resembles mammalian serine proteases a nd is structurally distinct from the papain superfamily of thiol prote ases. Given the interesting serine/cysteine protease hybrid nature of HAV 3C, we have probed its active site properties via the Raman spectr a of the acyl enzyme, 5-methylthiophene acryloyl HAV 3C, using the C24 S variant of the enzyme to obtain stoichiometric acylation. The Raman difference spectral data show that the major population of the acyl gr oups in the active site experiences electron polarization intermediate between that in the papain superfamily and that in a nonpolarizing si te. This is evidenced by the values of the acyl group ethylenic stretc hing frequency which occur near 1602 cm(-1) in a nonpolarizing environ ment, at 1588 cm(-1) when bound to HAV 3C (C24S), and at 1579 cm(-1) i n acyl papains, The value of the electronic absorption maximum for the HAV 3C (C24S) acyl enzyme and the deacylation rate constant fit the c orrelation developed for the papain superfamily, suggesting that for H AV 3C too, polarizing forces in the active site can contribute to rate acceleration via transition state stabilization. The major population in the active site is s-cis about the acyl group's C1-C2 bond, but th ere is a second population that is s-trans, and this secondary populat ion is not polarized. The two populations are evidenced by the presenc e of two sets of marker bands for s-cis and s-trans in the Raman spect ra, which occur principally in the C=C stretching region near 1600 cm( -1), in the C-C stretching region near 1100 cm(-1), and near 560 cm(-1 ). The positions of the acyl carbonyl features in the Raman spectra po int to hydrogen-bonding strengths of 20-25 kJ mol(-1) between the C=O and H-bonding donors in the active site, The 5-methylthiophene acryloy l HAV 3C (C24S) is a relatively unreactive acyl enzyme, deacylating wi th a pK(a) of 7.1 and a rate constant of 0.000 31 s(-1) at pH 9. Unlik e most other cysteine or serine protease acyl enzymes characterized by Raman spectroscopy, no changes in the Raman spectrum could be detecte d with changes in pH.