Nishida C R, de Montellano P R
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
J Biol Chem. 2001 Jun 8;276(23):20116-24. doi: 10.1074/jbc.M101548200. Epub 2001 Mar 22.
To clarify the role of the autoinhibitory insert in the endothelial (eNOS) and neuronal (nNOS) nitric-oxide synthases, the insert was excised from nNOS and chimeras with its reductase domain; the eNOS and nNOS inserts were swapped and put into the normally insertless inducible (iNOS) isoform and chimeras with the iNOS reductase domain; and an RRKRK sequence in the insert suggested by earlier peptide studies to be important (Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777) was mutated. Insertless nNOS required calmodulin (CaM) for normal NOS activity, but the Ca(2+) requirement for this activity was relaxed. Furthermore, insert deletion enhanced CaM-free electron transfer within nNOS and chimeras with the nNOS reductase, emphasizing the involvement of the insert in modulating electron transfer. Swapping the nNOS and eNOS inserts gave proteins with normal NOS activities, and the nNOS insert acted normally in raising the Ca(2+) dependence when placed in eNOS. Insertion of the eNOS insert into iNOS and chimeras with the iNOS reductase domain significantly lowered NOS activity, consistent with inhibition of electron transfer by the insert. Mutation of the eNOS RRKRK to an AAAAA sequence did not alter the eNOS Ca(2+) dependence but marginally inhibited electron transfer. The salt dependence suggests that the insert modulates electron transfer within the reductase domain prior to the heme/reductase interface. The results clarify the role of the reductase insert in modulating the Ca(2+) requirement, electron transfer rate, and overall activity of nNOS and eNOS.
为阐明自身抑制插入序列在内皮型一氧化氮合酶（eNOS）和神经型一氧化氮合酶（nNOS）中的作用，将该插入序列从nNOS及其与还原酶结构域的嵌合体中切除；交换eNOS和nNOS的插入序列，并将其放入通常无插入序列的诱导型一氧化氮合酶（iNOS）亚型及其与iNOS还原酶结构域的嵌合体中；此外，早期肽研究表明插入序列中的RRKRK序列很重要（萨勒诺，J.C.，哈里斯，D.E.，伊里扎里，K.，帕特尔，B.，莫拉莱斯，A.J.，史密斯，S.M.，马尔塔塞克，P.，罗曼，L.J.，马斯特斯，B.S.，琼斯，C.L.，魏斯曼，B.A.，莱恩，P.，刘，Q.，和格罗斯，S.S.（1997年）《生物化学杂志》272卷，29769 - 29777页），对其进行了突变。无插入序列的nNOS正常的一氧化氮合酶活性需要钙调蛋白（CaM），但该活性对Ca²⁺的需求有所放宽。此外，插入序列的缺失增强了nNOS及其与nNOS还原酶的嵌合体中无CaM时的电子传递，强调了插入序列在调节电子传递中的作用。交换nNOS和eNOS的插入序列得到了具有正常一氧化氮合酶活性的蛋白质，并且当nNOS插入序列置于eNOS中时，其在提高对Ca²⁺的依赖性方面正常发挥作用。将eNOS插入序列插入iNOS及其与iNOS还原酶结构域的嵌合体中显著降低了一氧化氮合酶活性，这与插入序列对电子传递的抑制作用一致。将eNOS的RRKRK突变为AAAAA序列并未改变eNOS对Ca²⁺的依赖性，但略微抑制了电子传递。盐依赖性表明插入序列在血红素/还原酶界面之前调节还原酶结构域内的电子传递。这些结果阐明了还原酶插入序列在调节nNOS和eNOS对Ca²⁺的需求、电子传递速率及总体活性方面的作用。
