Effect of selective chemical modification and CNBr-cleavage at methionine20 in catalytic activity and substrate binding properties of porcine pancreatic phospholipase A2.
Porcine pancreatic phospholipase A2 contains 2 methionine (Met) residues located at positions 8 and 20, respectively. Reaction of the enzyme with methyliodide and iodoacetic acid resulted in the selective methylation and carboxymethylation, respectively, of Met20. It was found that porcine pancreatic iso-phospholipase A2, possessing only Met8, was not affected by either modification. Reaction of porcine phospholipase A2 with cyanogen bromide in 0.1 N hydrochloric acid gave rise to cleavage only at Met20. The enhanced reactivity of Met20 compared to that of Met8 is in agreement with the known X-ray structure of phospholipase A2 which shows that Met8 is located in the interior of the protein, while Met20 is at the surface. Both methylation and carboxymethylation of Met20 do not significantly affect catalytic and substrate binding properties of the enzyme. In contrast, the more rigorous cleavage at Met20 by CNBr resulted in the loss of catalytic activity, while substrate and Ca2+ binding was diminished only to a limited extent. Most likely CNBr cleavage at Met20 perturbs the active site despite the fact that the N-terminal fragment Ala1-Hse20 is still bound via the disulfide bridge Cys11-Cys77 to the remainder of the protein. The results obtained strongly suggest that the conformation of the sequences Ala1-Hse20 and/or Asp21-Gly26 are important for the maintenance of the special microenvironment of the active site cleft.
van der Wiele
F C
FC
Laboratory of Biochemistry, State University of Utrecht, The Netherlands.
Dekker
N
N
Van Scharrenburg
G J
GJ
Hartskeerl
R A
RA
Meijer
H
H
De Haas
G H
GH
Van Deenen
L L
LL
Slotboom
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003488:Cyanogen Bromide; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity; D013552:Swine
D008715:Methionine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D003488:Cyanogen Bromide