Enzymatic activity of the SARS coronavirus main proteinase dimer.
The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH)(2)-Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K(m), and is unusually sensitive to ionic strength and reducing agents.
Graziano
Vito
V
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
McGrath
William J
WJ
DeGruccio
Ann Marie
AM
Dunn
John J
JJ
Mangel
Walter F
WF
D003001:Cloning, Molecular; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013696:Temperature
D010455:Peptides; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases