Site-directed Mutagenesis Of Gln49-PLA₂ And Study On Enzymatic Activity

A novel basic phospholipase A₂, named Gln49-PLA₂, is purified from Agkistrodonblomhoffii ussurensis snake venom. Myotoxic, cytotoxic and significant anticoagulantactivities have been detected, but devoid of hydrolysis activity of phospholipids. The mainpurpose of this study is to elucidate the reason and mechanism of catalytic inactivity ofGln49-PLA₂ and to further study its structure-function relationship.After bioinformatics analyzing and homology modeling, the main finding is that enzymaticinactivity of Gln49-PLA₂ is contributed to the replacement of Asp49 by Gln49, a residueconsidered to be a key component of the Ca²⁺ binding domains.To identify the structure-function relationship of the enzyme, the mutants ofGln49-PLA₂—fQ49D-PLA₂ and fQ49K-PLA₂ are expressed as inclusion body in E.coli, withpET-32a(+) vector, after site-directed mutagenesis of its code gene.By immobilized metal affinity chromatography (IMAC), the fusion protein is on-columnrefolded and purified from inclusion body particles. Under the optimized conditions: pH8.9Tris-HCl buffer, NaCl 0.5mol/L, imidazole 5mmol/L, GSH/GSSG 1:1 (1mmol/L), the highestyield of 6.889mg per liter culture is achieved, and the specific activity of fusion protein is36U/mg.The fQ49D-PLA₂ is cleaved by protease Factor Xa in vitro, and the mature Q49D-PLA₂ isobtained by further purification with Hitrap SP cation exchange and Superdex 75 columnschromatography. The results of enzymatic activity assay show that the specific activity of72U/mg is achieved for Q49D-PLA₂. The results demonstrate that the 49th glutamine aminoacid residue is the key of enzymatic inactivity of Gln49-PLA₂.Meanwhile, via the above three-step chromatography, Q49K-PLA₂ is obtained fromfQ49K-PLA₂ and no activity is detected.In summary, all these results contribute to the direct proof for the structure-functionrelationship of Gln49-PLA₂.