Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.
The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.
Janssen
M J
MJ
Department of Enzymology and Protein Engineering (CBLE, Institute of Biomembranes), Faculty of Chemistry, Utrecht University, PO Box 80. 054, 3508 TB Utrecht, The Netherlands.
van de Wiel
W A
WA
Beiboer
S H
SH
van Kampen
M D
MD
Verheij
H M
HM
Slotboom
A J
AJ
Egmond
M R
MR
D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D007850:Lauric Acids / Q000494:pharmacology; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics*; D054467:Phospholipases A2; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013552:Swine; D013816:Thermodynamics
D004791:Enzyme Inhibitors; D007850:Lauric Acids; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C066179:2-dodecanoylamino-1-hexanolphosphoglycol; D006639:Histidine; D010741:Phospholipases A; D054467:Phospholipases A2