Introduction of a C-terminal aromatic sequence from snake venom phospholipases A2 into the porcine pancreatic isozyme dramatically changes the interfacial kinetics.
Porcine pancreatic phospholipase A2 (PLA2) was modified by single and multiple site-directed mutations at sites thought to be involved in interfacial binding. Charged and polar residues in the C-terminal region were replaced by aromatic residues on the basis of an analogy with snake venom PLA2s, which display high affinity for a zwitterionic interface. The PLA2 variants constructed were N117W, N117W/D119Y and K116Y/N117W/D119Y. Titration with micelles of a zwitterionic substrate suggests that the variants N117W and K116Y/N117W/D119Y possess improved ability to bind to the micellar substrate interface, relative to the wild-type enzyme. Improved interfacial binding was confirmed by direct binding studies with micelles of a zwitterionic substrate analogue, indicating up to five times higher affinity for both variants. Interfacial binding is not improved for the variant N117W/D119Y. Maximal enzyme velocities (Vapp./max) with the zwitterionic substrate were between 25 and 75% of that of the wild-type enzyme. However, competitive inhibition and direct binding studies with a strong inhibitor revealed that the affinity for substrate present at the interface (Km*) is perturbed by the mutations made. For the variant N117W, the slight decrease observed in Vapp./max is most likely made up of a 24-fold reduction in catalytic turnover (kcat) and 18-fold improved substrate binding (Km*).
Janssen
M J
MJ
Department of Enzymology, Faculty of Chemistry, Utrecht University, The Netherlands.
Burghout
P J
PJ
Verheij
H M
HM
Slotboom
A J
AJ
Egmond
M R
MR
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D014644:Genetic Variation; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008823:Micelles; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012910:Snake Venoms / Q000378:metabolism*; D013552:Swine
D007527:Isoenzymes; D008823:Micelles; D011993:Recombinant Fusion Proteins; D012910:Snake Venoms; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium