Mutations on N-terminal region of Taiwan cobra phospholipase A(2) result in structurally distorted effects.
In the present study, three Taiwan cobra PLA(2) variants were prepared by adding an extra N-terminal Met, substituting Asn-1 by Met or deleting the N-terminal heptapeptide. Recombinant PLA(2) mutants were expressed in Escherichia coli (E. coli), and purified to homogeneity by reverse phase HPLC. Fluorescence measurement showed that the hydrophobic character of the catalytic site, the microenvironment of Trp residues and energy transfer from excited Trp to 8-anilinonaphthalene sulfonate (ANS) were affected by N-terminal mutations. An alteration in the structural flexibility of the active site was noted with the mutants lacking the N-terminal heptapeptide or with an extra N-terminal Met added as evidenced by the inability of the two variants to bind with Ba(2+). Moreover, modification of Lys residues and energy transfer within the protein-ANS complex revealed that the Ca(2+)-induced change in the global structure of PLA(2) was different from that in N-terminal variants. Together with the fact that an 'activation network' connects the N-terminus with the active site, our data suggest that mutagenesis on the N-terminal region affects directly the fine structure of the catalytic site, which subsequently transmits its influence in altering the structure outside the active site of PLA(2).
Chiou
Yi-Ling
YL
Institute of Biomedical Sciences, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Lin
Shinne-Ren
SR
Chang
Long-Sen
LS
D000818:Animals; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D004546:Elapid Venoms / Q000737:chemistry / Q000201:enzymology*; D017815:Elapidae; D004735:Energy Transfer; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes / Q000737:chemistry; D020869:Gene Expression Profiling; D014644:Genetic Variation; D009154:Mutation; D054467:Phospholipases A2 / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013624:Taiwan
D004546:Elapid Venoms; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D001464:Barium; D054467:Phospholipases A2; D002118:Calcium