Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5.
Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5.
复制标题
组氨酸 607 和组氨酸 643 为磷酸二酯酶 5 中的催化金属支持提供了重要的相互作用。
DOI：
10.1021/bi000392m
复制
发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Corbin,JD
中科院分区：
文献类型：
--
作者：
Francis,SH;Turko,IV;Grimes,KA;Corbin,JD
I类环核苷酸磷酸二酯酶（PDE）共享含有18个不变残基的催化结构域。在cGMP结合cGMP特异性PDE（PDE 5）中，我们先前发现，当在Mg 2+存在下进行测定时，其中9个点突变的kcat显著降低;其中7个位于我们先前鉴定的原型金属结合基序A和B（HX 3 HX nE）中。其中两个金属结合基序在PDE中的串联排列是新颖的，这些基序中的残基是否参与催化活性的金属载体是该领域的一个基本问题。该报告表明，His-607（A基序）或His-643（B基序）突变为丙氨酸显著降低了Mn 2+或Mg 2+对PDE催化的支持，但His-647在B基序中的突变或Glu在任一基序中的突变不会。H607 A和H643 A突变体在Mn ~（2+）支持下的最大催化速率远大于Mg ~（2+）支持下的最大催化速率; H603 A突变体的催化活性受到Mn ~（2+）和Mg ~（2+）的微弱支持。在H607 A和H643 A中，Mn ~（2+）和Mg ~（2+）的K_（as）增加，但Mn ~（2+）的影响比Mg ~（2+）大2倍。任何其他保守残基（Asn-604、Asp-644、His-675、Asp-714和Asp-754）的突变引起催化的Mn 2+或Mg 2+支持的不显著变化。这项研究确定了PDE 5中有助于与催化相关金属相互作用的特定残基。综合数据表明，尽管在PDE和某些金属内肽酶中的每个HX 3 HX nE基序之间具有高度的序列相似性，但PDE采用不同的残基互补与参与催化的金属相互作用。
Class I cyclic nucleotide phosphodiesterases (PDEs) share a catalytic domain containing 18 invariant residues. In cGMP-binding cGMP-specific PDE (PDE5), we showed previously that point mutation of nine of these profoundly decreaseskcatwhen the assay is conducted in the presence of Mg2+; seven of these are in the prototypical metal-binding motifs A and B (HX3HXnE) that we identified earlier. Tandem arrangement of two of these metal-binding motifs in PDEs is novel, and whether residues within these motifs are involved in metal support of catalytic activity is a fundamental question in this field. This report shows that mutation of either His-607 (A motif) or His-643 (B motif) to alanine profoundly diminishes support of PDE catalysis by Mn2+or Mg2+, but mutation of His-647 in B motif or of Glu in either motif does not. H607A and H643A mutants have much greater maximum catalytic rates supported by Mn2+than that by Mg2+; catalytic activity of H603A mutant is supported weakly by either. In H607A and H643A,Kas for Mn2+and Mg2+are increased, but the effect of Mn2+is 2-fold greater than that of Mg2+in each. Mutation of any of the other conserved residues (Asn-604, Asp-644, His-675, Asp-714, and Asp-754) causes unremarkable changes in Mn2+or Mg2+support of catalysis. This study identifies specific residues in PDE5 that contribute to interactions with catalytically relevant metals. The combined data suggest that despite a high degree of sequence similarity between each HX3HXnE motif in PDEs and certain metallo-endopeptidases, PDEs employ a distinct complement of residues for interacting with metals involved in catalysis.