Evidence for an essential lysyl residue in phospholipase D from Streptomyces sp. by modification with diethyl pyrocarbonate and pyridoxal 5-phosphate.
Diethyl pyrocarbonate inactivated phospholipase D from Streptomyces PMF with second-order rate constants of 0.7 M-1 s-1 at pH 6.1 or 222 M-1 s-1 at pH 8.3 and 25 degrees C, and modified 5 His residues per enzyme molecule. The His residues, however, were not essential for activity because: (a) the second-order rate constants for reaction of diethyl pyrocarbonate with the His residues of the enzyme, which were 1.4 M-1 s-1 at pH 6.1 or 7.2 M-1 s-1 at pH 8.3 and 25 degrees C, differed, both at low and high pH values, from the inactivation rates, and (b) the reversal of His modification by hydroxylamine was not accompanied by recovery of activity. As demonstrated by dinitrophenylation experiments carried out on the treated enzyme, diethyl pyrocarbonate also modified up to 20 Lys residues per enzyme molecule. Other amino acid residues and the conformation and hydrodynamic volume of the enzyme were not modified. The involvement of a Lys residue in enzyme activity was confirmed through experiments with pyridoxal 5-phosphate which inactivated phospholipase D, after NaBH4 reduction, with a second-order rate constant of 3.5 M-1 s-1 at pH 8.5 and 15 degrees C. The inactivation took place with concomitant modification of 4 Lys residues, only one of which was found to be essential using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1538). Dicaproyl phosphatidylcholine markedly protected the enzyme against inactivation by DEP or PLP, and this strongly suggests that the essential Lys residue is located in or near the substrate binding site.
Secundo
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CNR, Istituto di Chimica degli Ormoni, Milano, Italy.
Carrea
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D'Arrigo
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Servi
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D001665:Binding Sites; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013302:Streptomyces / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis
D013438:Sulfhydryl Compounds; D011732:Pyridoxal Phosphate; D004228:Dithionitrobenzoic Acid; D010739:Phospholipase D; D008239:Lysine; D004047:Diethyl Pyrocarbonate