Particulate methane monooxygenase (pMMO) is a membrane protein embedded in the intracytoplasmic membrane of methane-oxidizing bacteria. Structural analysis of pMMO showed the existence of a hydrophilic region exposed outside of the bacterial membrane. To obtain information regarding the role of this hydrophilic region in the enzymatic function of pMMO, trypsin proteolysis of the membrane-bound form of pMMO from Methylosinus trichosporium OB3b was performed at 4 °C. The polypeptides produced by this hydrolysis were analyzed by polyacrylamide gel electrophoresis and MALDI-TOF/TOF. Furthermore, the influence of this tryptic digestion on the methane hydroxylation and propene epoxidation enzymatic activities of pMMO was investigated. Among the three subunits of pMMO, PmoB and PmoC were hydrolyzed by trypsin, but PmoA was not. With 10 mg L⁻¹ trypsin, both terminal regions or the C-terminal region of PmoC polypeptide was selectively hydrolyzed. Furthermore, the stability of pMMO was decreased by this digestion. These results indicate that PmoC plays a role in maintaining the stability of pMMO in vitro. On the other hand, the digestion of PmoB with 100 mg L⁻¹ trypsin produced several polypeptides, indicating that trypsin digestion occurs at several sites of the hydrophilic region of PmoB. Hydrolysis led to a decrease in pMMO activity towards methane hydroxylation and propene epoxidation. These results indicate that the hydrophilic region of PmoB is critically important for the enzymatic function of pMMO, which is consistent with the models of the functional mechanism of pMMO proposed so far.

微粒甲烷单加氧酶 (pMMO) 是一种嵌入甲烷氧化细菌胞质内膜的膜蛋白。pMMO 的结构分析表明存在暴露在细菌膜外的亲水区域。为了获得有关该亲水区域在 pMMO 酶促功能中的作用的信息，胰蛋白酶对来自Methylosinus trichosporium的膜结合形式的 pMMO 进行蛋白酶解OB3b 在 4 °C 下进行。通过聚丙烯酰胺凝胶电泳和MALDI-TOF/TOF分析由该水解产生的多肽。此外，研究了这种胰蛋白酶消化对 pMMO 的甲烷羟基化和丙烯环氧化酶活性的影响。在 pMMO 的三个亚基中，PmoB 和 PmoC 被胰蛋白酶水解，而 PmoA 则没有。用10mg L ^-1胰蛋白酶选择性水解PmoC多肽的两个末端区域或C-末端区域。此外，这种消化降低了 pMMO 的稳定性。这些结果表明 PmoC 在维持 pMMO 体外稳定性方面发挥了作用。另一方面，PmoB 用 100 mg L ^-1胰蛋白酶产生了几种多肽，表明胰蛋白酶消化发生在 PmoB 亲水区域的几个位点。水解导致 pMMO 对甲烷羟基化和丙烯环氧化的活性降低。这些结果表明 PmoB 的亲水区域对 pMMO 的酶促功能至关重要，这与迄今为止提出的 pMMO 功能机制模型一致。