Semisynthesis of phospholipase A2. The effect of substitution of amino-acid residues at positions 6 and 7 in bovine and porcine pancreatic phospholipases A2 on catalytic and substrate-binding properties.
The N-terminal alpha-helical region of phospholipase A2 is an important part of the enzyme for catalytic activity and lipid binding. Porcine pancreatic phospholipase A2 has Arg-Ser at positions 6 and 7, whereas the bovine enzyme has Asn-Gly. To pursue further the effects of these variable residues on differences in enzymatic properties, we prepared and studied the following semisynthetic analogs of epsilon-amidinated phospholipase A2 (AMPA): porcine [Ala7]AMPA, [Gly7]AMPA, [Asn6]AMPA, [Asn6-Gly7]AMPA and bovine [Ser7]AMPA and [Arg6-Ser7]AMPA. As we had previously found for the Asn6 leads to Arg bovine substitution, an Asn6-Gly7 leads to Arg5-Ser7 bovine substitution similarly improves the catalytic activity, the affinity for neutral lipid-water interfaces and the capacity to penetrate lecithin monolayers, while just changing Gly7 leads to Ser produces almost no effect on these properties. Ser7 leads to Ala and Ser7 leads to Gly substitutions in porcine AMPA did not affect penetration or lipid binding, although they did diminish catalytic activity (which is true of all substitutions made in the porcine enzyme). Arg6 leads to Asn substitution in porcine AMPA decreases penetration of lecithin monolayers, but not as much as it was improved by the Asn6 leads to Arg substitution in bovine AMPA. In contrast to the dramatic increase in affinity for lipid-water interfaces of Asn6 leads to Arg substitution in bovine AMPA, no decrease in affinity was found for Arg6 leads to Asn substitution in porcine AMPA. This difference is most likely due to the fact that the porcine enzyme has positively charged Lys and His in place of the Lys10, Glu17 pair that lie very close to residue 6 in the bovine structure. It can thus be conclude that (with the exception of Gly7 leads to Ser in bovine AMPA) all the substitutions tried at positions 6 and 7 in bovine and porcine AMPAs have definite effects on the catalytic activity.
D000577:Amides; D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D050356:Lipid Metabolism; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
D000577:Amides; D000596:Amino Acids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2