Characterization of the dimer-monomer equilibrium of the papaya Copper/Zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation.
The coding region of the copper/zinc superoxide dismutase (Cu/Zn SOD) cDNA from papaya fruit, Carica papaya L. cv. Tainong 2, was cloned into an expression vector, pET-20b(+). The Cu/Zn SOD was expressed in Escherichia coli and purified by His-tag technique. Two active forms of the enzyme (30% dimer and 70% monomer) in equilibrium were observed. The activity of the dimeric enzyme was higher than that of the monomeric form. The thermal inactivation rate constant K(d) values calculated for the dimer and monomer at 90 degrees C were -0.0203 and -0.0216 min(-1), and the half-lives for inactivation were 41.9 and 31.8 min, respectively. This indicated that the dimeric enzyme was more stable than its monomeric form. The dimerization of the enzyme was inhibited under acidic pH (below 3.0) or imidazole buffer (above 0.5 M), whereas it was not affected under alkaline pH (above 9.0). Both activity and forms of the enzyme were not affected by 1-4% SDS. Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin or chymotrypsin. In addition, mutation of the papaya Cu/Zn SOD at position 48 from Leu to Phe (L48F) affected the association of monomer, whereas a mutant with Lys substitution (L48K) at the same position tended to dissociate into monomeric form.
Lin
C T
CT
Institute of Marine Biotechnology, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 2024, Taiwan, Republic of China. ctlin@ntou66.ntou.edu.tw
Kuo
T J
TJ
Shaw
J F
JF
Kao
M C
MC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers; D019281:Dimerization; D005638:Fruit / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D013482:Superoxide Dismutase