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 PM F 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 p er 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 rat es, and (b) the reversal of His modification by hydroxylamine was not accompanied by recovery of activity. As demonstrated by dinitrophenyla tion experiments carried out on the treated enzyme, diethyl pyrocarbon ate also modified up to 20 Lys residues per enzyme molecule. Other ami no acid residues and the conformation and hydrodynamic volume of the e nzyme were not modified. The involvement of a Lys residue in enzyme ac tivity was confirmed through experiments with pyridoxal 5-phosphate wh ich 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 residu es, only one of which was found to be essential using the kinetic meth od of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1538). Dicaproyl pho sphatidylcholine 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.