Modification of Arginine Residues in Porcine Pancreatic Phospholipase A2

Abstract

Although phenylglyoxal monohydrate reacts with Arg-6 in porcine pancreatic phospholipase Az, concomittantly the a-amino group of the N-terminal Ala-1 residue is quantitatively transaminated. Due to this latter reaction the enzymatic activity toward micellar substrate is lost irrespective of the Arg-6 modification. Upon reaction of [7-'4C]phenylglyoxal monohydrate with a-amino-blocked phospholipase Az analogs, two molecules of the reagent were incorporated per protein molecule, which were found to be present on Arg-6. Removal of a-amino-blocking groups after the modification reaction furnished the corresponding Arg-6-modified phospholipases possessing 30-38 "/, of their original specific enzymatic activities in the egg-yolk assay.After reaction of 1,2-[l-'4C]cyclohexanedione with porcine phospholipase A2 the crude reaction mixture was purified by chromatography on quaternary diethyl-(2-hydroxypropyl)aminoethyl-Sephadex in the presence of borate. A fraction was obtained containing a pure protein in which one molecule of ''C-labeled reagent per protein molecule was incorporated which was found to be localized almost exclusively on Arg-6. Cyclohexanedione modification of Arg-6 in phospholipase A2 does not significantly influence its catalytic activity when assayed toward monomeric and micellar substrates. The results of direct binding experiments using substrate analogs and of monolayer studies of the phospholipase modified at Arg-6 by cyclohexanedione are in agreement with previous findings that Arg-6 is involved in the interaction of the enzyme with lipid-water interfaces.Phospholipase Az has been isolated from many sources [1,2]. It is a small ( M , about 14000) water-soluble protein, catalyzing the hydrolysis of 3-sn-phosphoglycerides, whereas 1 -sn-phosphoglycerides are competitive inhibitors [3]. The enzyme has an absolute requirement of Ca" and reaches its highest activity when bound to aggregated substrates [4]. It has been shown that the snake venom enzymes bind Caz+ and substrates via an ordered mechanism in which Ca2+ is bound first [ 5 ] , while for porcine pancreatic enzyme a random mechanism has been proposed [4,6]. Investigations of the binding of pancreatic phospholipase Az to aggregated substrate and substrate analogs, have led to the proposition of a micelle-binding site on the porcine enzyme, which is topologically distinct from the active centre [7,8]. For this binding site, the term 'interface recognition site' has been proposed and indications were found for an important role of the N-terminal part of the protein in the formation of the interface recognition site [8 -101. Furthermore, it was found that the Arg-6/Ser-7 bond was protected from trypsine cleavage when porcine phospholipase AZ was bound to micelles [lo], strongly suggesting that Argd belongs to the interface recognition site. To further elaborate the role of Arg-6 in the protein, an arginine-modification study was started. Takahashi [I11 and Borders and Riordan [I21 have shown the applicability of vicinal diketons in arginine modifi...

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