Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A₂ on catalytic properties

Abstract

Residue 31 of porcine pancreatic phospholipase A2 (PLA2) is located at the entrance to the active site. To study the role of residue 31 in PLA2, six mutant enzymes were produced by site-directed mutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Ser or Gly. Direct binding studies indicated a three to six times greater affinity of the Trp31 PLA2 for both monomeric and micellar substrate analogs, relative to the wild-type enzyme. The other five mutants possess an unchanged affinity for monomers of the product analog n-decylphosphocholine and for micelles of the diacyl substrate analog rac-1,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine. The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholine were decreased 9-20 times for these five mutants. Kinetic studies with monomeric substrates showed that the mutants have Vmax values which range between 15 and 70% relative to the wild-type enzyme. The Vmax values for micelles of the zwitterionic substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3-50 times. The Km values for the monomeric substrate and the Km values for the micellar substrate were hardly affected in the case of five of the six mutants, but were considerably decreased when Trp was present at position 31. The results of these investigations point to a versatile role for the residue at position 31: involvement in the binding and orientating of monomeric substrate (analogs), involvement in the binding of the enzyme to micellar substrate analogs and possibly involvement in shielding the active site from excess water.

Sequences, geographic variations and molecular phylogeny of venom phospholipases and threefinger toxins of eastern India Bungarus fasciatus and kinetic analyses of its Pro31 phospholipases A2

“…In fact, many of the inactive Lys49 PLAs from crotalid venoms also contain Pro31 [32], while other viperid venom PLAs usually contain Trp31 [17,32]. Group IA or elapid venom PLAs with higher catalytic activities usually contain Lys, Arg or Leu at position 31 [33,34]. Previous studies of pancreatic PLA mutants revealed that replacements of Leu31 or Arg31 by other amino acids reduced the enzymatic activities considerably [34,35].…”

Interfacial Recognition by Bee Venom Phospholipase A2: Insights into Nonelectrostatic Molecular Determinants by Charge Reversal Mutagenesis

“…Results presented here affirm that tight binding of PLA2 to anionic interfaces must come from multiple weak interactions; however, the task is to identify the factors that contribute toward these interactions. On the basis of the results at hand, it appears that hydrophobic (46)(47)(48), electrostatic (15,19,49,50), and hydrogen-bonding (51) interactions are implicated (16,17). The task of dissecting such contributions is daunting because they are interdependent (one may contribute to the other).…”

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli

“…The fusion protein was S-sulfonated (Tannhauser and Scheraga, 1985), precipitated by dilution with acetic acid and lyophilized. Reoxidation, trypsin activation and purification of the porcine pancreatic [Met8+Leu, Met20+Leu]APLA2 was performed as described elsewhere (Kuipers et al, 1990b). With the Ssulfonated Cro-LacZ -HP-PLA, fusion protein, two routes were attempted to obtain biosynthetic PLA,.…”