Holzman T F, Baldwin T O
Proc Natl Acad Sci U S A. 1980 Nov;77(11):6363-7. doi: 10.1073/pnas.77.11.6363.
Upon limited proteolysis of luciferases from the luminous marine bacteria Photobacterium fischeri, Photobacterium phosphoreum, and Beneckea harveyi, the rate of loss of luciferase activity is the same as the rate of loss of the heavier subunit of all three enzymes. It thus appears that the larger subunit of the luciferase from P. phosphoreum should be designated alpha based on its apparent homology with the alpha subunits of the luciferases from B. harveyi and P. fischeri. The luciferase from B. harveyi is more sensitive to chymotrypsin than to trypsin; the luciferases of the Photobacterium species are more sensitive to trypsin than to chymotrypsin. Proteolytic inactivation of all three luciferases results from hydrolysis of a few peptide bonds in the alpha subunit; the proteolytic fragments from the three luciferases in 0.50 M phosphate are approximately the same size, indicating that the three enzymes have a protease-labile region at about the same position in the primary structure of their alpha subunits. Phosphate stabilizes all three luciferases against inactivation by proteases. Formation and degradation of intermediate species derived from the alpha subunits are readily observable in all three luciferases. Phosphate alters both the rate of product formation and the sites of peptide bond scission. The beta subunits of the luciferases from the two Photobacterium species, unlike the enzyme of B. harveyi, appear to be degraded in buffers containing low concentrations of phosphate; in high-phosphate buffers, the beta subunits of all three luciferases appear to resist proteases. Analysis of native and chymotrypsin-inactivated P. fischeri and P. phosphoreum luciferases in the analytical ultracentrifuge indicates that, as with B. harveyi luciferase, the products of limited proteolysis do not dissociate under nondenaturing conditions. The fact that the luciferases from evolutionarily diverse species of luminous bacteria have protease-sensitive bonds in the same region of the alpha subunit that are stabilized by anions strongly suggests that the protease-labile region of the alpha subunit is either an integral component of or in close proximity to the active center.
对发光海洋细菌费氏发光杆菌、磷光发光杆菌和哈维贝内克氏菌的荧光素酶进行有限的蛋白酶解时，三种酶的荧光素酶活性丧失速率与较重亚基的丧失速率相同。因此，基于磷光发光杆菌荧光素酶的较大亚基与哈维贝内克氏菌和费氏发光杆菌荧光素酶的α亚基具有明显的同源性，该较大亚基应被指定为α亚基。哈维贝内克氏菌的荧光素酶对胰凝乳蛋白酶比对胰蛋白酶更敏感；发光杆菌属的荧光素酶对胰蛋白酶比对胰凝乳蛋白酶更敏感。三种荧光素酶的蛋白酶解失活是由α亚基中少数肽键的水解引起的；在0.50M磷酸盐中，三种荧光素酶的蛋白酶解片段大小大致相同，表明这三种酶在其α亚基一级结构的大致相同位置有一个对蛋白酶敏感的区域。磷酸盐可稳定所有三种荧光素酶，使其免受蛋白酶的失活作用。在所有三种荧光素酶中都很容易观察到源自α亚基的中间物种的形成和降解。磷酸盐会改变产物形成的速率以及肽键断裂的位点。与哈维贝内克氏菌的酶不同，两种发光杆菌属荧光素酶的β亚基似乎在含有低浓度磷酸盐的缓冲液中被降解；在高磷酸盐缓冲液中，所有三种荧光素酶的β亚基似乎都能抵抗蛋白酶。在分析超速离心机中对天然和经胰凝乳蛋白酶失活的费氏发光杆菌和磷光发光杆菌荧光素酶进行分析表明，与哈维贝内克氏菌荧光素酶一样，有限蛋白酶解的产物在非变性条件下不会解离。来自进化上不同的发光细菌物种的荧光素酶在α亚基的同一区域具有对蛋白酶敏感的键且被阴离子稳定，这一事实强烈表明α亚基的蛋白酶敏感区域要么是活性中心的一个组成部分，要么与活性中心紧密相邻。
