Ikeda K, Inoue S, Amasaki C, Teshima K, Ikezawa H
Department of Biochemistry, Osaka University of Pharmaceutical Sciences.
J Biochem. 1991 Jul;110(1):88-95. doi: 10.1093/oxfordjournals.jbchem.a123547.
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)
从蜡样芽孢杆菌IAM 1208菌株的培养物中分离出了水解磷脂酰胆碱的磷脂酶C，即所谓的“磷脂酶C”（PLC）。纯化酶的氨基酸组成和部分N端序列与ATCC 10987菌株PLC的核苷酸序列预期结果高度一致[约翰森等人（1988年），《基因》65卷，293 - 304页]。在25℃、pH 8.０、离子强度0.2且存在饱和量Zn²⁺（0.1 mM）的条件下，采用pH计测定法研究了PLC催化单分散短链磷脂酰胆碱（二氰基磷脂酰胆碱，N = 3 - 6）水解时动力学参数对链长的依赖性。将结果与蛇毒磷脂酶A2的结果进行了比较[寺岛等人（1989年），《生物化学杂志》106卷，518 - 527页]。发现PLC与底物分子头部基团的相互作用对结合非常重要。在相同条件下还研究了单分散二C5PC以及二C16PC与 Triton X - 100混合胶束水解时动力学参数的pH依赖性。发现一个可电离基团，其pK值因底物结合从7.77变为8.30，对催化至关重要。根据X射线晶体学和化学修饰研究结果[霍夫等人（1989年），《自然》338卷，357 - 360页；利特尔（1977年），《生物化学杂志》167卷，399 - 404页]，该基团初步被确定为His 14。（摘要截取自250字）
