Krook M, Ghosh D, Strömberg R, Carlquist M, Jörnvall H
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):502-6. doi: 10.1073/pnas.90.2.502.
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.
从人胎盘中纯化出两种不同形式的单体烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）连接的前列腺素脱氢酶/羰基还原酶，结果表明它们因一个赖氨酸残基的修饰而有所不同。修饰型和未修饰型蛋白质以大约1:3的比例可重复回收，且两者化学性质稳定。修饰型更偏酸性（pI约为7.4，而未修饰型pI约为7.7），但在特异性和活性方面与未修饰型无法区分。氨基酸分析、序列分析、质谱分析和化学合成确定修饰后的残基为N6-(1-羧乙基)赖氨酸，丙酸的C-2连接到赖氨酸-238的侧链N上。该化合物可由赖氨酸残基和丙酮酸通过席夫碱及随后的还原反应形成。该酶及其依赖烟酰胺腺嘌呤二核苷酸（NAD(+)）的对应物亲缘关系较远（残基一致性为23%），并且在短链脱氢酶家族中属于同一类别。对3α/20β-羟基类固醇脱氢酶三级结构的比对和模型构建表明，羰基还原酶有一个额外的环（第149 - 189位），形成一个单独的延伸部分并取代了C末端的主链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。第238位的羧乙基赖氨酸对应于几种短链脱氢酶中对蛋白水解敏感的位置，在模型中定义不太明确，但靠近表面，并且与观察到的可及性和酶特性相符。
