Hwang Y, Catalano C E, Feiss M
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.
Biochemistry. 1996 Feb 27;35(8):2796-803. doi: 10.1021/bi952322z.
Terminase the DNA packaging enzyme of bacteriophage chi, is a heteromultimer of gpNul (21 kDa) and gpA (74 kDa) subunits, encoded by the chi Nul and A genes, respectively. Sequence comparisons indicate that both gpNu1 and gpA have a match to the P-loop motif of ATPase centers, which is a glycine-rich segment followed by a lysine. By site-specific mutagenesis, we changed the lysines of the putative P-loops of gpNul (k35) and gpA (K497) to arginine, alanine, or aspartic acid, and studied the mutant enzymes by kinetic analysis and photochemical cross-linking with 8-azido-ATP. Both the gpNul and gpA subunits of wild-type terminase were covalently modified with 8-N3[32P] ATP in the presence of UV light. Saturation occurred with apparent dissociation constants of 508 and 3.5 microM for gpNul and gpA, resepctively. ATPase assays showed two activities: a low-affinity activity (Km=469 microM), and a high-affinity activity (Km=4.6 microM). The gpNul K35A and gpNul K35D mutant terminases showed decreased activity in the low-affinity ATPase activity. The reduced activities of these enzymes were recovered when 10 times more DNA was added, suggesting that the primary defect of the enzymes is alteration of the nonspecific, double-stranded DNA binding activity of terminase. ATPase assays and photolabeling of the gpA K497A and gpA K497D mutant terminases showed reduced affinity for ATP at the high-affinity site which was not restored by increased DNA. In summary, the results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-affinity site in gpA.
噬菌体χ的DNA包装酶——末端酶，是由gpNul（21 kDa）和gpA（74 kDa）亚基组成的异源多聚体，分别由χ Nul和A基因编码。序列比较表明，gpNu1和gpA均与ATP酶中心的P环基序匹配，该基序是一个富含甘氨酸的片段，后面跟着一个赖氨酸。通过定点诱变，我们将gpNul（K35）和gpA（K497）推定的P环中的赖氨酸分别替换为精氨酸、丙氨酸或天冬氨酸，并通过动力学分析以及与8-叠氮基-ATP的光化学交联研究了突变酶。在紫外线存在下，野生型末端酶的gpNul和gpA亚基均与8-N3[32P]ATP发生共价修饰。gpNul和gpA的表观解离常数分别为508和3.5 microM时达到饱和。ATP酶分析显示出两种活性：低亲和力活性（Km = 469 microM）和高亲和力活性（Km = 4.6 microM）。gpNul K35A和gpNul K35D突变型末端酶在低亲和力ATP酶活性中表现出活性降低。当添加10倍以上的DNA时，这些酶降低的活性得以恢复，这表明这些酶的主要缺陷是末端酶非特异性双链DNA结合活性的改变。gpA K497A和gpA K497D突变型末端酶的ATP酶分析和光标记显示，其在高亲和力位点对ATP的亲和力降低，增加DNA不能恢复这种亲和力。总之，结果表明gpNul中存在低亲和力、DNA刺激的ATP酶中心，gpA中存在高亲和力位点。
