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  • Title: Structural importance of the amino-terminal residue of pancreatic phospholipase A2.
    Author: van Scharrenburg GJ, Jansen EH, Egmond MR, de Haas GH, Slotboom AJ.
    Journal: Biochemistry; 1984 Dec 04; 23(25):6285-94. PubMed ID: 6441599.
    Abstract:
    To study the structural importance of the NH2-terminal Ala1 residue of pancreatic phospholipase A2, several mutants were prepared by a stepwise semisynthetic approach. 13C NMR spectroscopy of 90%-enriched [[3-13C]Ala1] phospholipases A2 shows the pK values of the alpha-NH3+ groups of porcine enzyme, porcine isoenzyme, bovine enzyme, and equine enzyme to be 8.4, 8.8, 8.9, and 8.8, respectively. A group titrating with a pK of approximately 6.3, present only in the porcine and equine phospholipases A2, presumably originating from Glu71, disappears in the presence of 0.2 M Ca2+, while the pK values of their alpha-NH3+ groups shift to 9.3 and 9.0, respectively. No such effects were observed for the porcine isoenzyme and bovine enzyme, which lack an acidic side chain in position 71. It can thus be concluded that the equine phospholipase A2, like the porcine enzyme, possesses in addition to the catalytic Ca2+ binding site also a second, low-affinity Ca2+ binding site, which is not present in the porcine isophospholipase A2 and bovine phospholipase A2. From the titration behavior of the alpha-NH3+ group of equine and porcine [[3-13C]-Ala1]phospholipases A2 in the presence of micelles of n-hexadecylphosphocholine, it seems very likely that at alkaline pH the equine phospholipase A2, like the porcine enzyme, requires the second Ca2+ ion for optimal binding to neutral lipid-water interfaces. Semisynthetic porcine phospholipase A2 analogues in which the position of the alpha-NH3+ group is varied have lost their affinity toward neutral lipid-water interfaces and consequently their catalytic activity on micellar substrates. Most of these phospholipase A2 analogues retain, however, some of their enzymatic activity on monomeric substrate. Substitution of the side chain of Ala1 in porcine phospholipase A2 by hydrophobic side chains abolishes almost all activity due to the loss of affinity for neutral lipid-water interfaces. In contrast, NH2-terminal residues having more polar side chains affect only slightly phospholipase A2 activity. Compared to the native enzyme, these latter phospholipase A2 analogues show an increased penetration capacity for monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Probably, the interaction of hydrophobic amino acid residues at the 1-position with other hydrophobic side chains present in their vicinity prevents the correct positioning of the alpha-NH3+ group, thereby leading to loss of catalytic activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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