title: Amino acid substitutions of the NH2-terminal Ala1 of porcine pancreatic phospholipase A2: a monolayer study.
abstract: Previously it has been shown that the binding of porcine pancreatic phospholipase A2 to lipid-water interfaces is governed by the pK of the alpha-NH3+ group of the N-terminal alanine. Chemically modified phospholipases A2 in which the N-terminal Ala has been replaced by D-Ala or in which the polypeptide chain has been elongated with DL-Ala no longer display activity toward micellar substrate. The activity of DL-Ala-1-, [D-Ala1]-, and [Gly1]phospholipases A2 on substrate monolayers, which allow a continuous change in the packing density of the lipid molecule, was investigated. At pH 6 [Gly1]phospholipase A2 behaves like the native enzyme on lecithin monolayers. DL-Ala1- and [D-Ala1]phospholipases A2, although they are active in this system, showed a weaker lipid penetration capacity at this pH. Studies on the pH and Ca2+ ion dependency of the pre-steady-state kinetics and of the activity of these radiolabeled proteins showed that [D-Ala1]phospholipase A2 does not possess a second low-affinity site for Ca2+ ions in contrast to the native phospholipase A2. This second low-affinity Ca2+ binding site, which is also absent in [Gly1]phospholipase A2, is induced in the latter enzyme by the presence of lipid-water interfaces.
mesh_terms: D000409:Alanine; D000818:Animals; D007700:Kinetics; D008567:Membranes, Artificial; D008823:Micelles; D010179:Pancreas / Q000201:enzymology; D010740:Phospholipases / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011312:Pressure; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013499:Surface Properties; D013552:Swine
chemical_list: D008567:Membranes, Artificial; D008823:Micelles; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D000409:Alanine