Effect of tryptophan insertions on the properties of the human group IIA phospholipase A2: mutagenesis produces an enzyme with characteristics similar to those of the human group V phospholipase A2.
An important characteristic of the human group IIA secreted phospholipase A(2) (IIA PLA(2)) is the extremely low activity of this enzyme with phosphatidylcholine (PC) vesicles, mammalian cell membranes, and serum lipoproteins. This characteristic is reflected in the lack of ability of this enzyme to bind productively to zwitterionic interfaces. Part of the molecular basis for this lack of activity is an absence of tryptophan, a residue with a known preference for residing in the interfacial region of zwitterionic phospholipid bilayers. In this paper we have replaced the eight residues that make up the hydrophobic collar on the interfacial binding surface of the enzyme with tryptophan. The catalytic and interfacial binding properties of these mutants have been investigated, particularly those properties associated with binding to and hydrolysis of zwitterionic interfaces. Only the insertion of a tryptophan at position 3 or 31 produces mutants that significantly enhance the activity of the human IIA enzyme against zwitterionic interfaces and intact cell membranes. Importantly, the ability of the enzyme mutants to hydrolyze PC-rich interfaces such as the outer plasma membrane of mammalian cells was paralleled by enhanced interfacial binding to zwitterionic interfaces. The corresponding double tryptophan mutant (V3,31W) displays a specific activity on PC vesicles comparable to that of the human group V sPLA2. This enhanced activity includes the ability to interact with human embryonic kidney HEK293 cells, previously reported for the group V enzyme [Kim, Y. J., Kim, K. P., Rhee, H. J., Das, S., Rafter, J. D., Oh, Y. S., and Cho, W. (2002) J. Biol. Chem. 277, 9358-9365].
Beers
Stephen A
SA
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, UK.
Buckland
Andrew G
AG
Giles
Niroshini
N
Gelb
Michael H
MH
Wilton
David C
DC
D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002942:Circular Dichroism; D017815:Elapidae; D054501:Group II Phospholipases A2; D054505:Group V Phospholipases A2; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding / Q000235:genetics; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010713:Phosphatidylcholines; D014364:Tryptophan; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054505:Group V Phospholipases A2; D054467:Phospholipases A2; C101424:phospholipase A2-V, Trimeresurus