Active site properties of the 3C proteinase from hepatitis A virus (a hybrid 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 and is structurally distinct from the papain superfamily of thiol proteases. Given the interesting serine/cysteine protease hybrid nature of HAV 3C, we have probed its active site properties via the Raman spectra of the acyl enzyme, 5-methylthiophene acryloyl HAV 3C, using the C24S variant of the enzyme to obtain stoichiometric acylation. The Raman difference spectral data show that the major population of the acyl groups in the active site experiences electron polarization intermediate between that in the papain superfamily and that in a nonpolarizing site. This is evidenced by the values of the acyl group ethylenic stretching frequency which occur near 1602 cm(-1) in a nonpolarizing environment, at 1588 cm(-1) when bound to HAV 3C (C24S), and at 1579 cm(-1) in acyl papains. The value of the electronic absorption maximum for the HAV 3C (C24S) acyl enzyme and the deacylation rate constant fit the correlation developed for the papain superfamily, suggesting that for HAV 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 there is a second population that is s-trans, and this secondary population is not polarized. The two populations are evidenced by the presence of two sets of marker bands for s-cis and s-trans in the Raman spectra, 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 point 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 acryloyl HAV 3C (C24S) is a relatively unreactive acyl enzyme, deacylating with a pKa of 7.1 and a rate constant of 0.000 31 s(-1) at pH 9. Unlike most other cysteine or serine protease acyl enzymes characterized by Raman spectroscopy, no changes in the Raman spectrum could be detected with changes in pH.
Dinakarpandian
D
D
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Shenoy
B
B
Pusztai-Carey
M
M
Malcolm
B A
BA
Carey
P R
PR
D000086042:3C Viral Proteases; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006507:Hepatovirus / Q000201:enzymology*; D008958:Models, Molecular; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D014764:Viral Proteins
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases