Tanaka M, Sato K, Uchida T
J Biol Chem. 1981 Nov 25;256(22):11397-400.
Substrate specificity of a prolyl hydroxylase from Vinca rosea suspension-cultured cells was studied using synthetic oligo(L-proline)s and their t-butyloxycarbonyl derivatives (Pron and Boc-Pron; n = 2-8) as peptidyl substrates. All peptides with a residual number of 5 or greater served as substrates in the enzyme reaction at 30 degrees C, after the preincubation of the enzyme and peptides at 0 degrees C prior to addition of cofactors and cosubstrate. Under the same conditions, the hydroxylation of Pro5 reached a plateau within 10 min, but that of Boc-Pro8 and poly(L-proline) increased linearly up to 40 min. If the preincubation temperature was raised to 30 degrees C, only Pro5 among the peptides was unable to serve as a substrate. The optimum temperature of the enzyme was 30 degrees C toward Boc-Pro8 and poly(L-proline) but it decreased to 15 degrees C using Pro5. These data suggest that the enzyme can bind Pro5 only at low temperature. Poly(L-proline) and Boc-Pron (n greater than or equal to 5) in aqueous solution are known to have a left-handed helical structure (poly(L-proline) II helix). Moreover, Pro5 was indicated as forming this helix below 10 degrees C. Accordingly, the enzyme recognizes the poly(L-proline) II helix, that is, the secondary structure of a substrate rather than the primary structure.
利用合成的寡聚（L-脯氨酸）及其叔丁氧羰基衍生物（Pron和Boc-Pron；n = 2 - 8）作为肽基底物，研究了长春花悬浮培养细胞中脯氨酰羟化酶的底物特异性。在添加辅因子和共底物之前，将酶和肽在0℃预孵育后，所有残基数为5或更多的肽在30℃的酶反应中都可作为底物。在相同条件下，Pro5的羟化在10分钟内达到平稳状态，但Boc-Pro8和聚（L-脯氨酸）的羟化在40分钟内呈线性增加。如果将预孵育温度提高到30℃，肽中只有Pro5不能作为底物。该酶对Boc-Pro8和聚（L-脯氨酸）的最适温度为30℃，但使用Pro5时最适温度降至15℃。这些数据表明该酶仅在低温下能结合Pro5。已知聚（L-脯氨酸）和水溶液中的Boc-Pron（n≥5）具有左手螺旋结构（聚（L-脯氨酸）II螺旋）。此外，Pro5在10℃以下被表明形成这种螺旋。因此，该酶识别聚（L-脯氨酸）II螺旋，即底物的二级结构而非一级结构。
