Roles of aromatic residues in high interfacial activity of Naja naja atra phospholipase A2.
Acidic phospholipase A2 (PLA2) from the venom of Chinese cobra (Naja naja atra) has high activity on zwitterionic membranes and contains six aromatic residues, including Tyr-3, Trp-18, Trp-19, Trp-61, Phe-64, and Tyr-110, on its putative interfacial binding surface. To assess the roles of these aromatic residues in the interfacial catalysis of N. n. atra PLA2, we mutated them to Ala and measured the effects on its interfacial catalysis. Enzymatic activities of the mutants toward various vesicle substrates and human neutrophils indicate that all but Trp-18 make significant contributions to interfacial catalysis. Among these aromatic residues, Trp-19, Trp-61, and Phe-64 play the most important roles. Binding affinities of the mutants for phospholipid-coated beads and their monolayer penetration indicate that Trp-19, Trp-61, and Phe-64 are critically involved in interfacial binding of N. n. atra PLA2 and penetrate into the membrane during the interfacial catalysis of N. n. atra PLA2. Further thermodynamic analysis suggests that the side chain of Phe-64 is fully inserted into the hydrophobic core of membrane whereas those of Trp-19 and Trp-61 are located in the membrane-water interface. Together, these results show that all three types of aromatic residues can play important roles in interfacial binding of PLA2 depending on their location and side-chain orientation. They also indicate that these aromatic side chains interact with membranes in distinct modes because of their different intrinsic preference for different parts of membranes.
Sumandea
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Department of Chemistry (M/C 111), University of Illinois at Chicago 60607-7061, USA.
Das
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Sumandea
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Cho
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W
D000595:Amino Acid Sequence; D000598:Amino Acids, Cyclic / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004546:Elapid Venoms / Q000096:biosynthesis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010742:Phospholipid Ethers / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D000598:Amino Acids, Cyclic; D004546:Elapid Venoms; D008081:Liposomes; D010742:Phospholipid Ethers; D010743:Phospholipids; D011108:Polymers; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D008734:Methoxyhydroxyphenylglycol; D014364:Tryptophan; D010741:Phospholipases A; D054467:Phospholipases A2; C010117:3,4-dihydroxyphenylglycol