Activation of human rhinovirus-14 3C protease

The catalytic efficiency of human rhinovirus-14 (HRV14) 3C protease as a fu nction of solvents and other regulators has been investigated using synthet ic peptides as substrates. The proteolytic activity of HRV14 3C was found t o be strongly stimulated by a series of anions in vitro and the activation was accompanied by changed Km, kcat. and increased kcat/Km values. A more t han 72-fold increase in the 3C catalytic efficiency toward peptide substrat es was observed in the presence of 0.8 M sodium sulfate. Several approaches , including size-exclusion chromatography and chemical cross-linking experi ments, suggested that no oligomerization of the sc enzyme occurred in the p resence of activating anions. However, the anions did induce a significant conformational change of HRV14 3C protease, as revealed by circular dichroi sm spectrometry and tyrosine fluorescence analyses, which might contribute to 3C enzyme activation. Finally. the results obtained from 3C protease inh ibitor studies suggested that the S1 specificity pocket of HRV14 sc was mai nly affected by the activating anions. An induced-fit catalysis mechanism f or viral proteases is discussed. (C) 2001 Academic Press.