Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2.
A free N-terminal alpha-NH3+ group is absolutely required for full catalytic activity of phospholipase A2 on aggregated substrates. To elucidate how this alpha-NH3+ group triggers catalytic activity, we specifically transaminated this group in various pancreatic phospholipases A2. Porcine, porcine iso-, equine, human, ovine, and bovine phospholipases A2 all loose catalytic activity on micellar substrates due to the inability of the transaminated proteins to bind to neutral micellar substrate analogues, as was found for the zymogens. Loss of activity is pseudo first order, the rate constants being different for the enzymes studied. The transaminated phospholipases A2 have an intact active site, as catalytic activities on monomeric substrates are comparable to those of the respective zymogens. The X-ray structure of transaminated bovine phospholipase A2 at 2.1-A resolution shows that the N-terminal region and the sequence 63-72 in this protein are more flexible than in the native enzyme. Also, in this respect, the transaminated enzyme very much resembles the zymogen structure. In good agreement with this, it was found by photochemically induced dynamic nuclear polarization 1H NMR that aromatic resonances of Trp-3 and Tyr-69 are affected by transamination. In addition, fluorescence spectroscopy of the unique Trp-3 in transaminated bovine phospholipase A2 revealed a red shift of the emission maximum indicative of a more polar environment of Trp-3 in the transaminated phospholipase A2 as compared to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002417:Cattle; D006736:Horses; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D011487:Protein Conformation; D012756:Sheep; D013045:Species Specificity; D013379:Substrate Specificity; D013552:Swine; D014961:X-Ray Diffraction
D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2