Proton-Nuclear-Magnetic-Resonance/pH-Titration Studies of the Histidines of Pancreatic Phospholipase A2

Abstract

The study by means of 'H nuclear magnetic resonance (NMR) of the histidines of phospholipase A2 isolated from porcine, bovine and equine pancreas is reported. Assignment of the histidine resonances was achieved by comparison of different enzymes and the use of paramagnetic probes. pH titration curves for various histidyl resonances were obtained and compared in the presence and absence of calcium. Calcium is shown to lower the pKa of the active site histidine. The NMR results are compared with the known X-ray three-dimensional structure for the bovine enzyme.Phospholipase A2 is an enzyme, present in high concentrations in snake and bee venoms and in mammalian pancreas, which catalyses the hydrolysis of the fatty acid ester bond at the 2-position of 3-snphosphoglycerides [l]. The enzyme is secreted by the pancreas as a zymogen and zymogens from a number of mammals have been purified. The amino acid sequences of porcine, bovine and equine pancreatic phospholipase A2 and of the corresponding zymogens have been determined [2-41. From the porcine pancreas an isoproenzyme occurring in a 5:95 ratio compared with the more abundant zymogen was isolated [5] and its amino acid sequence determined [6]. The zymogens are converted into the active enzymes by the action of trypsin which splits a peptide bond Arg-Ala with the removal of the N-terminal heptapeptide from the molecule [7]. In Table 1 the primary structures of equine, bovine and porcine phospholipases are shown. The three-dimensional structure and disulfide bond connections of the bovine enzyme have recently been elucidated [8].Inactivation studies using p-bromophenacyl bromide [9] have shown that His-48 is involved in the active site of phospholipase A2. This active site was also found to pre-exist for a main part already in the zymogen and, in fact, both zymogen and enzyme hydrolyse monomeric substrates at comparable rates. The rate of hydrolysis is dramatically increased in the case of the enzyme if the substrate is in micellar form [lo]. Calcium ions are specifi-Abbreviarions NMR, nuclear magnetic resonance; ppm, partsEnzyme Phosphohpase A2 (EC 3 1 1 4).per million cally required for catalytic activity and bind in a 1 : l molar ratio to the active enzyme and to the zymogen with similar affinities and producing similar conformational changes. His-48 was shown to be located very close to the metal ion binding site Proton nuclear magnetic resonance is the technique of choice for the study of proteins in detail in aqueous solution. In general, the low-field or aromatic region of a protein 'H NMR spectrum is the easiest to interpret because, apart from the exchangeable protons, only histidyl, tyrosyl, phenylalanyl and tryptophanyl residues give resonances in that region. The exchangeable protons can be removed by working in 2H20. The application of this technique to obtain knowledge of the environments and pKa values of histidyl groups in various proteins has been particularly successful due to the fact that the C-2 proton resonances of histidyl residues normally li...

Automated proton titrations of pancreatic phospholipase A2

“…[5], resulting in the values given in Table 2. Table 2 gives the average apparent and intrinsic pK values for the histidines, which are in excellent agreement with the 'H NMR values of Aguiar et al (10) and with the proton-titration results of Janssen et al (8) for the porcine enzyme.…”

Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: Absence of the conserved structural water

“…In the WT enzyme, the H2 of His-48 displays a pK, of 5.7 (Aguiar et al, 1979). In D99N, adistinct resonance, possibly aris ing from the H2 of the His-48, does not display a clear chemicalshift change from pH 8.9 (8.463 ppm) to 4.7 (8.462 ppm).…”

The amino acid sequence of the phospholipase A2 isoenzyme from porcine pancreas

“…The isoenzyme has been used for chemical modification of the single Met residue [5] and the absence of Met 2° and His 17 has made possible an unequivocal assignment of the 1H-NMR resonances of the histidine residues 17, 48 and 115 and methionine residues 8 and 20 found in the major porcine pancreatic enzyme [6]. In this paper we describe the chemical and enzymatic fragmentation of porcine isophospholipase and the isolation of the peptides.…”