Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.
Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.
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蛋白质中的羧乙基赖氨酸：天然羰基还原酶/NADP( )依赖性前列腺素脱氢酶。
DOI：
10.1073/pnas.90.2.502
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发表时间：
1993
影响因子：
11.1
通讯作者：
Jörnvall,H
中科院分区：
文献类型：
--
作者：
Krook,M;Ghosh,D;Strömberg,R;Carlquist,M;Jörnvall,H
从人胎盘中纯化了两种不同形式的单体NADP（+）-连接的前列腺素脱氢酶/羰基还原酶，并通过赖氨酸残基的修饰显示出不同。修饰的和未修饰的蛋白质以约1：3的比例可重复地回收，并且两者都是化学稳定的。修饰形式的酸性更强（pI约为7.4，pI约为7.7），但在特异性和活性方面与未修饰形式无区别。氨基酸分析、序列分析、质谱和化学合成鉴定出修饰后的残基为N6-（1-羧乙基）赖氨酸，其中丙酸的C-2连接在Lys-238的侧链N上。该化合物可以由赖氨酸残基和丙酮酸盐通过席夫碱和随后的还原形成。该酶及其NAD（+）依赖性对应物是远亲（23%残基同一性），并具有相同的短链脱氢酶家族分配。3 α/20 β-羟基类固醇脱氢酶三级结构的比对和模型构建表明，羰基还原酶具有额外的环（位置149-189），其形成单独的延伸并取代主链C-末端β链。这种变化影响了底物口袋，解释了不同的底物特异性，但保留了已知功能重要性的残基。位置238处的羧乙基赖氨酸对应于几种短链淀粉酶中的蛋白水解敏感位置，在模型中不太明确，但靠近表面，并且与观察到的可及性和酶性质相容。
Two different forms of the monomeric NADP(+)-linked prostaglandin dehydrogenase/carbonyl reductase were purified from human placenta and shown to differ by the modification of a lysine residue. The modified and the unmodified proteins were reproducibly recovered in a ratio of approximately 1:3, and both were chemically stable. The modified form was more acidic (pI approximately 7.4 versus pI approximately 7.7) but indistinguishable from the unmodified form in specificity and activity. Amino acid analysis, sequence analysis, mass spectrometry, and chemical synthesis identified the modified residue as N6-(1-carboxyethyl)lysine with C-2 of propionic acid attached to the side-chain N of Lys-238. This compound can be formed from the lysine residue and pyruvate via a Schiff base and subsequent reduction. The enzyme and its NAD(+)-dependent counterpart are distantly related (23% residue identity) and have the same family assignment to short-chain dehydrogenases. Alignments and model-building into the tertiary structure of 3 alpha/20 beta-hydroxysteroid dehydrogenase show that carbonyl reductase has an extra loop (positions 149-189) that forms a separate extension and replaces a backbone C-terminal beta-strand. This change affects the substrate pocket, explaining the different substrate specificities but conserves residues of known functional importance. Carboxyethyllysine at position 238 corresponds to a proteolysis-sensitive position in several short-chain dehydrogenases, less well-defined in the model but close to a surface, and is compatible with the accessibility and enzyme properties observed.