The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130 → Tyr almost completely inhibited enzyme activity at 37 °C; activity was, however, partially restored by the following exchanges: Ser-27 → Pro, His-135 → Arg or His-137 → Arg. To investigate this phenotypic reversion, 2A proteinases with the mutations Phe-130 → Tyr, Phe-130 → Tyr/His-135 → Arg, Phe-130 → Tyr/His-137 → Arg, His-135 → Arg or His-137 → Arg were expressed in Escherichia coli and purified. None of these mutations affected the affinity of the enzyme for a peptide substrate. However, the temperature-dependence of enzyme activity, as assayed by cleavage of a peptide substrate and by monitoring the toxicity of the proteinases towards the E. coli strain BL21(DE3), and the structural stability, as monitored by 8-anilino-1-naphthalenesulphonic acid fluorescence and CD spectrometry, were affected. The thermal transition temperatures for both the activity and the stability of the Phe-130 → Tyr 2A proteinase were reduced by about 17 °C compared with the wild-type enzyme. The presence of the additional mutations His-135 → Arg or His-137 → Arg in the Phe-130 → Tyr mutant increased temperature stability by 3 °C and 6 °C respectively. Thus essential interactions exist within the C-terminal domain of human rhinoviral 2A proteinases which contribute to the overall stability and integrity of the enzyme.

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