Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily.
Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily.
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DOI：
10.1042/bj3440047
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发表时间：
1999-11
期刊：
影响因子：
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通讯作者：
M. Bowker-Kinley;K. M. Popov
中科院分区：
文献类型：
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作者：
M. Bowker-Kinley;K. M. Popov
本研究采用定点突变的方法，对丙酮酸脱氢酶激酶的Asn-247、Asp-282、Gly-284、Gly-286和Gly-319的功能进行了研究。重组激酶，野生型，Asn-247 Ala，Asp-282 Ala，Gly-284 Ala，Gly-286 Ala和Gly-319 Ala，在细菌中表达，纯化，并表征。Asn-247 Ala、Asp-282 Ala和Gly-286 Ala三种突变激酶缺乏任何明显的活性。另外两个突变体Gly-284 Ala和Gly-319 Ala具有催化活性，其表观V（max）值接近野生型激酶（分别为67和85与70 nmol/min/mg）。Gly-319 Ala对核苷酸底物的表观K（m）值显著增加（1500对16 μ M）。相比之下，Gly-284 Ala的K（m）值仅略高于野生型酶（28对16微M）。ATP结合分析表明，Asn-247 Ala、Asp-282 Ala和Gly-286 Ala不能与核苷酸结合。Gly-284 Ala的K（d）值略高于野生型酶（分别为7和4 μ M）。与动力学分析一致，Gly-319 Ala突变体结合ATP如此之差，以至于难以确定结合常数。尽管Asn-247 Ala、Asp-282 Ala和Gly-286 Ala缺乏酶活性，但它们仍然能够结合蛋白质底物，如它们在与野生型激酶的竞争测定中的负显性效应所示。CD旋光分析结果表明，Asp-282 Ala和Gly-286 Ala的二级结构没有发生较大变化。这些结果强烈表明，丙酮酸脱氢酶激酶的催化结构域位于C-末端。此外，该催化结构域可能与ATP酶/激酶超家族成员[分子伴侣热休克蛋白90（Hsp 90）、DNA促旋酶B和组氨酸蛋白激酶]的催化结构域类似地折叠。
In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent V(max) values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent K(m) value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 microM). In contrast, Gly-284Ala had only a slightly higher K(m) value than the wild-type enzyme (28 versus 16 microM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The K(d) value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 microM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].