Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.
Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.
复制标题
大豆脂氧合酶-1第二配位球突变体的结构和功能表征。
DOI：
10.1021/bi002893d
复制
发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Holman,TR
中科院分区：
文献类型：
--
作者：
Tomchick,DR;Phan,P;Cymborowski,M;Minor,W;Holman,TR
Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln495and Gln697, which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn694). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln495. The nature of the C−H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH-is the active site base. A new transition state model for hydrogen atom abstraction is proposed.
