Human rhinovirus 2A proteinase mutant and its second-site revertants.
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 degrees 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-I-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 degrees 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 degrees C and 6 degrees 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.
Luderer-Gmach
M
M
Institut für Biochemie der Universitaet Wien, Vienna, Austria.
Liebig
H D
HD
Sommergruber
W
W
Voss
T
T
Fessl
F
F
Skern
T
T
Kuechler
E
E
D001665:Binding Sites; D002942:Circular Dichroism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D005820:Genetic Testing; D006801:Humans; D007700:Kinetics; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012229:Rhinovirus / Q000201:enzymology*; D013696:Temperature; D014170:Transformation, Genetic; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus