Lee S H, Seo H Y, Kim J C, Heo W D, Chung W S, Lee K J, Kim M C, Cheong Y H, Choi J Y, Lim C O, Cho M J
Department of Biochemistry, Gyeongsang National University, Chinju 660-701, Korea.
J Biol Chem. 1997 Apr 4;272(14):9252-9. doi: 10.1074/jbc.272.14.9252.
NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.
NAD激酶是一种依赖Ca2+/钙调蛋白（CaM）的酶，能够将细胞内的NAD转化为NADP。从豌豆幼苗中纯化得到的该酶可被高度保守的大豆CaM即SCaM-1激活，但不能被不同的大豆CaM异构体SCaM-4激活（Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806 - 21812）。为了确定哪些结构域导致了NAD激酶的这种差异激活，通过在SCaM-4和SCaM-1之间交换功能结构域，构建了一系列嵌合SCaM。SCaM-4111是一种包含SCaM-4第一个结构域的嵌合SCaM-1，其激活NAD激酶的能力严重受损（仅为最大激活能力的40%）。SCaM-1444是一种包含SCaM-1第一个结构域的嵌合SCaM-4，它对NAD激酶的激活接近完全（约70%）。只有包含SCaM-1结构域I的嵌合体对NAD激酶的激活大于最大激活能力的一半。为了确定结构域I中负责这种差异激活的氨基酸残基，构建并测试了SCaM-1的七个单残基取代突变体对NAD激酶的激活情况。在这些突变体中，只有K30E和G40D表现出NAD激酶激活能力大幅降低。此外，一个包含这两个突变的双残基取代突变体K30E/G40D，其激活NAD激酶的潜力严重受损，仅达到最大激活能力的20%。此外，一个三突变体K30E/M36I/G40D完全消除了NAD激酶的激活。因此，我们的数据表明，CaM的结构域I在SCaM-1和SCaM-4对NAD激酶的差异激活中起关键作用。此外，SCaM-1的赖氨酸30和谷氨酸40残基对该功能至关重要。
