Lin A N, Ashley G W, Stubbe J
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Biochemistry. 1987 Nov 3;26(22):6905-9. doi: 10.1021/bi00396a006.
The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.
（1）用二硫苏糖醇对酶进行预还原；（2）在没有外源还原剂的情况下用底物处理对氧化还原活性硫醇进行特异性氧化；（3）用碘乙酰胺对其他硫醇进行烷基化；（4）用二硫苏糖醇还原二硫键并用[1-14C]碘乙酰胺进行烷基化。经二硫苏糖醇还原的大肠杆菌B1亚基能够将3当量的CDP转化为dCDP，并被5.4当量的14C标记。胰蛋白酶肽段测序表明，2.8当量的14C位于B1 C末端的半胱氨酸-752和-757上，而1.0 - 1.5当量的14C位于半胱氨酸-222和-227上。因此，似乎有两组氧化还原活性二硫醇参与底物还原。莱氏乳杆菌还原酶能够将1.1当量的CTP转化为dCTP，并被2.1当量的14C标记。胰蛋白酶肽段测序表明，1.4当量的14C位于C-E-G-G-A-C-P-I-K的两个半胱氨酸上。该肽段与大肠杆菌B1 C末端肽段C-E-S-G-A-C-K-I的含硫醇区域表现出显著且意外的相似性。
