Kinetics of the hydrolysis of monodispersed and micellar phosphatidylcholines catalyzed by a phospholipase C from Bacillus cereus.
The phosphatidylcholine-hydrolyzing phospholipase C, so-called "phospholipase C" (PLC), was isolated from the culture of Bacillus cereus strain IAM 1208. The amino-acid composition and partial N-terminal sequence of the purified enzyme were in good agreement with those expected from the nucleotide sequence for a PLC of strain ATCC 10987 [Johansen et al. (1988) Gene 65, 293-304]. The chain-length dependence of kinetic parameters for the PLC-catalyzed hydrolysis of monodispersed short-chain phosphatidylcholines (diCNPC, N = 3-6) was studied by a pH-stat assay method at 25 degrees C, pH 8.0, and ionic strength 0.2 in the presence of saturating amounts of Zn2+ (0.1 mM). The result was compared with those for snake venom phospholipases A2 [Teshima et al. (1989) J. Biochem. 106, 518-527]. It was found that the interaction of the PLC with the head group of the substrate molecule is very important for the binding. The pH dependences of kinetic parameters for the hydrolysis of monodispersed diC5PC and mixed micelles of diC16PC with Triton X-100 were also studied under the same conditions. An ionizable group, whose pK value is perturbed from 7.77 to 8.30 by substrate binding, was found to be essential to the catalysis. This group was tentatively assigned to His 14 on the basis of the results on X-ray crystallographic and chemical modification studies [Hough et al. (1989) Nature 338, 357-360 and Little (1977) Biochem. J. 167, 399-404].(ABSTRACT TRUNCATED AT 250 WORDS)
Ikeda
K
K
Department of Biochemistry, Osaka University of Pharmaceutical Sciences.
Inoue
S
S
Amasaki
C
C
Teshima
K
K
Ikezawa
H
H
D000596:Amino Acids / Q000032:analysis; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008823:Micelles; D010713:Phosphatidylcholines / Q000378:metabolism*; D010738:Type C Phospholipases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D008823:Micelles; D010713:Phosphatidylcholines; D010738:Type C Phospholipases