Jiang P, Ananthanarayanan V S
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
J Biol Chem. 1991 Dec 5;266(34):22960-7.
An attempt has been made to understand the conformational determinants that govern the hydroxylation of selected lysyl residues in the nascent collagen molecule by lysyl hydroxylase (EC 1.14.11.4). A series of peptide substrates of the enzyme, ranging in length from 3 to 12 residues, were synthesized. These included: tert-butyloxylcarbonyl (t-Boc)-Ile-Lys-Gly; Boc-Ala-Lys-Gly; N-acetyl-Ala-Lys-Gly-Ser; Hyp-Gly-Pro-Lys-Gly-Glu; Leu-Hyp-Gly-Ala-Lys-Gly-Glu; Gly-Phe-Hyp-Gly-Leu-Hyp-Gly-Ala-Lys-Gly-Glu; (Hyp-Gly-Pro-Lys-Gly-Glu)2; and Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly. The conformational features of these peptides were studied by spectroscopic methods so as to relate this information with the kinetic parameters for the interaction of these peptides with purified lysyl hydroxylase. Spectroscopic data, supported by conformational energy calculations, indicated that the tripeptides t-Boc-Ile-Lys-Gly and t-Boc-Ala-Lys-Gly adopt a gamma-turn structure in water and trifluoroethanol with Lys in the second position of the turn. In the tetra- and larger peptides two structures, the beta-turn and a polyproline-II (PP-II) type extended conformation, were identified. The proportions of these two structures in a given peptide depended on the polarity of the solvent. All of the peptides were hydroxylated by lysyl hydroxylase isolated from chicken embryos. In contrast, a control peptide, t-Boc-Ala-Gly-Lys which adopted a beta-turn with Lys at the end of the turn, was not hydroxylated. Competitive inhibition of the hydroxylation of protocollagen by some of the peptides showed a common binding site for these substrates in the enzyme's active site. Kinetic data on the peptides indicated improved hydroxylation rate (higher Vmax) in peptides having relatively higher beta-turn content and improved binding (lower Km) in peptides with higher content of the PP-II structure. The efficacy of the substrate was also governed by its chain length. These data suggest that the conformational criterion for lysine hydroxylation in collagen-related peptides is the presence of a "bent" structure, such as the gamma- or beta-turn at the catalytic site of lysyl hydroxylase and an "extended" PP-II type structure at the binding site(s) of the enzyme's active site. This suggestion also provides a conformational rationale for earlier observations on the substrate specificity of lysyl hydroxylase.
人们试图了解控制赖氨酰羟化酶（EC 1.14.11.4）对新生胶原蛋白分子中选定赖氨酰残基进行羟化作用的构象决定因素。合成了一系列该酶的肽底物，长度从3个残基到12个残基不等。这些底物包括：叔丁氧羰基（t - Boc）-异亮氨酸 - 赖氨酸 - 甘氨酸；Boc - 丙氨酸 - 赖氨酸 - 甘氨酸；N - 乙酰 - 丙氨酸 - 赖氨酸 - 甘氨酸 - 丝氨酸；羟脯氨酸 - 甘氨酸 - 脯氨酸 - 赖氨酸 - 甘氨酸 - 谷氨酸；亮氨酸 - 羟脯氨酸 - 甘氨酸 - 丙氨酸 - 赖氨酸 - 甘氨酸 - 谷氨酸；甘氨酸 - 苯丙氨酸 - 羟脯氨酸 - 甘氨酸 - 亮氨酸 - 羟脯氨酸 - 甘氨酸 - 丙氨酸 - 赖氨酸 - 甘氨酸 - 谷氨酸；（羟脯氨酸 - 甘氨酸 - 脯氨酸 - 赖氨酸 - 甘氨酸 - 谷氨酸）₂；以及丙氨酸 - 精氨酸 - 甘氨酸 - 异亮氨酸 - 赖氨酸 - 甘氨酸 - 异亮氨酸 - 精氨酸 - 甘氨酸 - 苯丙氨酸 - 丝氨酸 - 甘氨酸。通过光谱方法研究了这些肽的构象特征，以便将这些信息与这些肽与纯化的赖氨酰羟化酶相互作用的动力学参数联系起来。光谱数据在构象能量计算的支持下表明，三肽t - Boc - 异亮氨酸 - 赖氨酸 - 甘氨酸和t - Boc - 丙氨酸 - 赖氨酸 - 甘氨酸在水和三氟乙醇中采用γ - 转角结构，赖氨酸位于转角的第二个位置。在四肽及更大的肽中，鉴定出了两种结构，即β - 转角和多聚脯氨酸II（PP - II）型伸展构象。给定肽中这两种结构的比例取决于溶剂的极性。所有这些肽都被从鸡胚中分离出的赖氨酰羟化酶羟化。相比之下，一种对照肽t - Boc - 丙氨酸 - 甘氨酸 - 赖氨酸，它在转角末端采用β - 转角结构且赖氨酸位于末端，未被羟化。一些肽对原胶原蛋白羟化的竞争性抑制表明这些底物在酶的活性位点有一个共同的结合位点。关于这些肽的动力学数据表明，β - 转角含量相对较高的肽中羟化速率提高（Vmax更高），而PP - II结构含量较高的肽中结合能力提高（Km更低）。底物的效力也受其链长的影响。这些数据表明，胶原蛋白相关肽中赖氨酸羟化的构象标准是存在一种“弯曲”结构，例如在赖氨酰羟化酶催化位点处的γ - 或β - 转角，以及在酶活性位点结合位点处的“伸展”PP - II型结构。这一观点也为早期关于赖氨酰羟化酶底物特异性的观察提供了构象依据。
