Volwerk J J, Shashidhar M S, Kuppe A, Griffith O H
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Biochemistry. 1990 Sep 4;29(35):8056-62. doi: 10.1021/bi00487a010.
The inositol phosphate products formed during the cleavage of phosphatidylinositol by phosphatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are 0.41, 3.62, 4.45, and 16.30 ppm for phosphatidylinositol, myo-inositol 1-monophosphate, myo-inositol 2-monophosphate, and myo-inositol 1,2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions this phospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myo-inositol 1,2-cyclic monophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myo-inositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myo-inositol 1,2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidyl-inositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phosphodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.
利用31P核磁共振（NMR）分析了蜡样芽孢杆菌的磷脂酰肌醇特异性磷脂酶C在裂解磷脂酰肌醇过程中形成的肌醇磷酸产物。31P NMR光谱可区分肌醇磷酸种类和磷脂酰肌醇。观察到的化学位移值（相对于磷酸），磷脂酰肌醇为0.41 ppm、肌醇1-单磷酸为3.62 ppm、肌醇2-单磷酸为4.45 ppm、肌醇1,2-环单磷酸为16.30 ppm。结果表明，在各种实验条件下，该磷脂酶C通过分子内磷酸转移反应裂解磷脂酰肌醇，生成二酰基甘油和D-肌醇1,2-环单磷酸。我们还报告了一项新的意外发现，即蜡样芽孢杆菌的磷脂酰肌醇特异性磷脂酶C能够水解肌醇环磷酸酯形成D-肌醇1-单磷酸。因此，该酶具有磷酸转移酶和环磷酸二酯酶活性。通过31P NMR观察到，第二个反应需要千倍更高的酶浓度。该反应具有区域特异性，因为只生成1-磷酸，也具有立体特异性，因为只有D-肌醇1,2-环单磷酸被水解。用针对蜡样芽孢杆菌磷脂酶C的单克隆抗体进行抑制实验表明，环磷酸二酯酶活性是该细菌酶所固有的。我们提出了蜡样芽孢杆菌磷脂酰肌醇特异性磷脂酶C的两步机制，涉及依次的磷酸转移酶和环磷酸二酯酶活性。该机制与著名的胰腺核糖核酸酶RNase A的两步机制相似。
