Acylation of porcine pancreatic phospholipase A2 influences penetration and substrate head-group binding, depending on the position of the acylated lysine in the enzyme molecule.
A porcine pancreatic phospholipase A2 mutant was constructed in which all nine lysines were replaced by arginines. The mutant displayed 68% residual activity on micellar zwitterionic substrates, indicating that lysines are not absolutely required for the catalytic action of the enzyme. Likewise, mutants with one single lysine present either at position 56, located close to the entrance of the active site, or at position 108, remote from the active site, were constructed. Selective acylation of Lys56 with acyl chains of two, eight or fourteen carbon atoms resulted in increased activities on 1,2-dioctanoylglycero-3-phosphocholine micelles. Moreover, acylation strongly influenced the affinity for these micelles, as was evidenced by an up to 60-fold increase in apparent Km. The kinetic properties of the (acylated) mutants were studied with the monolayer technique. Pre-steady-state kinetics showed that penetration into monomolecular layers composed of 1,2-didodecanoylglycero-3-phosphocholine was faster for acylated Lys56 derivatives than for non-acylated enzyme. The acylated enzymes were also capable of penetrating densely packed lipid films. This effect increased with increasing acyl chain length. The observed velocities in the steady state were similar for acylated and non-acylated Lys56 mutants. In contrast, no changes in the kinetic properties were observed after acylation of Lys108, located on the posterior part of the protein. Therefore, the effects observed upon acylation of Lys56 are probably specific. Apart from an increase in hydrophobicity, acylation of Lys results in charge neutralization. The latter effect was studied with a mutant in which Gln instead of Lys was present at position 56. The activity of this mutant on micellar substrates is higher than that of the parent Lys56, whereas its affinity for micelles is slightly improved. Therefore, whereas the charge at position 56 mainly influences the activity, the hydrophobicity of the introduced acyl chain mainly determines the affinity for aggregated lipids.
Lugtigheid
R B
RB
Department of Enzymology and Protein Engineering, C. B. L. E., Utrecht, The Netherlands.
Nicolaes
G A
GA
Veldhuizen
E J
EJ
Slotboom
A J
AJ
Verheij
H M
HM
De Haas
G H
GH
D000215:Acylation; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D013379:Substrate Specificity; D013552:Swine
D009838:Oligodeoxyribonucleotides; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine