Sharov V S, Schöneich C
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA.
Free Radic Biol Med. 2000 Nov 15;29(10):986-94. doi: 10.1016/s0891-5849(00)00400-7.
Recent studies have shown that the "calcium-sensor" protein calmodulin (CaM) suffers an age-dependent oxidation of methionine (Met) to methionine sulfoxide (MetSO) in vivo. However, MetSO did not accumulate on the Met residues that show the highest solvent-exposure. Hence, the pattern of Met oxidation in vivo may give hints as to which reactive oxygen species and oxidation mechanisms participate in the oxidation of this important protein. Here, we have exposed CaM under a series of different reaction conditions (pH, [Ca(2+)], [KCl]) to various biologically relevant reactive oxygen species and oxidizing systems (peroxides, HOCl, peroxynitrite, singlet oxygen, metal-catalyzed oxidation, and peroxidase-catalyzed oxidation) to investigate whether one of these systems would lead to an oxidation pattern of CaM similar to that observed in vivo. However, generally, these oxidizing conditions led to a preferred or exclusive oxidation of the C-terminal Met residues, in contrast to the oxidation pattern of CaM observed in vivo. Hence, none of the employed oxidizing conditions was able to mimic the age-dependent oxidation of CaM in vivo, indicating that other, yet unidentified oxidation mechanisms may be important in vivo. Some oxidizing species showed a quite-remarkable diastereoselectivity for the formation of either L-Met-D-SO or L-Met-L-SO. Diastereoselectivity was dependent on the nature of the oxidizing species but was less a function of the location of the target Met residue in the protein. In contrast, diastereoselective reduction of L-Met-D-SO by protein methionine sulfoxide reductase (pMSR) was efficient regardless of the position of the L-Met-D-SO residue in the protein and the presence or absence of calcium. With only the L-Met-D-SO diastereomer being a substrate for pMSR, any preferred formation of L-Met-L-SO in vivo may cause the accumulation of MetSO unless the oxidized protein is substrate for (accelerated) protein turnover.
最近的研究表明，“钙传感器”蛋白钙调蛋白（CaM）在体内会发生蛋氨酸（Met）随年龄增长被氧化为甲硫氨酸亚砜（MetSO）的现象。然而，MetSO并未在溶剂暴露程度最高的Met残基上积累。因此，体内Met氧化的模式可能会提示哪些活性氧物种和氧化机制参与了这种重要蛋白质的氧化过程。在此，我们将CaM置于一系列不同的反应条件（pH值、[Ca(2+)]、[KCl]）下，使其与各种生物学相关的活性氧物种和氧化系统（过氧化物、HOCl、过氧亚硝酸盐、单线态氧、金属催化氧化和过氧化物酶催化氧化）发生反应，以研究这些系统中的某一个是否会导致CaM出现与体内观察到的氧化模式相似的情况。然而，总体而言，这些氧化条件导致C末端Met残基优先或唯一被氧化，这与体内观察到的CaM氧化模式相反。因此，所采用的氧化条件均无法模拟CaM在体内随年龄增长的氧化过程，这表明其他尚未明确的氧化机制在体内可能很重要。一些氧化物种对L-Met-D-SO或L-Met-L-SO的形成表现出相当显著的非对映选择性。非对映选择性取决于氧化物种的性质，但较少取决于蛋白质中目标Met残基的位置。相比之下，蛋白质甲硫氨酸亚砜还原酶（pMSR）对L-Met-D-SO的非对映选择性还原无论L-Met-D-SO残基在蛋白质中的位置以及钙的存在与否都是有效的。由于只有L-Met-D-SO非对映异构体是pMSR的底物，体内L-Met-L-SO的任何优先形成都可能导致MetSO的积累，除非氧化的蛋白质是（加速）蛋白质周转的底物。
