Evidence for functional involvement of asparagine 67 in substrate recognition by snake venom phospholipases A2.
Site-directed mutagenesis studies of recombinant Trimeresurus flavoviridis venom gland phospholipase A2 (PLA2) showed that the Asn residue at position 67 takes part in recognition of the substrate 2-arachidonoyl sn-glycero-3-phosphocholine in both monomeric and micellar states. The amount of arachidonate released from phosphatidylcholine mixed micelles was reduced to 30% for N67D and N67K mutants, and to 70% for N67G mutant, but remained unchanged for N67S mutant. In contrast, for monomeric substrate, the activity was decreased to 40% for N67D and N67G and to 60% for N67K but remained unchanged for N67S. These results suggest that the properties of the side chain of residue 67 exert a significant influence on recognition of 2-arachidonoyl sn-glycero-3-phosphocholine.
Ogawa
T
T
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka. ogawa@biochem.tohoku.ac.jp
Shimohigashi
Y
Y
Ohno
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008066:Lipolysis / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D003435:Crotalid Venoms; D010713:Phosphatidylcholines; C029748:Trimeresurus venoms; C043644:1-stearoyl-2-arachidonylphosphatidylcholine; D001216:Asparagine; D010741:Phospholipases A; D054467:Phospholipases A2