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...