The mechanism of cis-trans isomerization of prolyl peptides by cyclophilin
The mechanism of cis-trans isomerization of prolyl peptides by cyclophilin
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DOI：
10.1021/ja020222s
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发表时间：
2002-06-26
影响因子：
15
通讯作者：
Bruice, TC
中科院分区：
文献类型：
--
作者：
Hur, S;Bruice, TC
通过对过渡态 (TS) 以及基态 (GS) 的顺式和反式形式 (GS) 的分子动力学 (MD) 模拟（当与 CyP 结合时和游离于水溶液中时），通过计算研究了亲环蛋白 (CyP) 催化的脯氨酰肽顺反异构化机制。 MD 模拟包括四种酶结合的四肽（Suc-Ala-XC(=O)-NPro-Phe-pNA；X = Gly、Trp、Ala 和 Leu）。在水中，脯氨酰酰胺键有利地是平面的，并且存在表现出C-N二面体的1,201扭曲的构象异构体。在活性位点，Asn102 的顺式脯氨酰胺羰基 0 和主链酰胺 N-H 之间的氢键保留了 C-N 二面体的 201 扭曲。 TS结构的特点是酰胺C-N键扭曲900度，并且由于Asn102(HN)和酰胺羰基O之间的距离较短，因此与Asn102更有利的相互作用。顺式-TS的构象变化还涉及酰胺N的金字塔化，这导致酰胺N和Arg55的胍基之间形成氢键。 Asn102 和 Arg55 在 CyP.cis 异构体中的位置与在 CyP.TS 中的位置相同。在无配体的 CyP 中，Arg55 胍基基团高度混乱，并且 Asn102 与配体结合的 CyP 中的位置相比被移位 1 埃。因此，Arg55 和 Asn102 的组织发生在底物结合时。组织酶结构与 TS 结构的几何互补性是由于与 GS 相比，TS 的脯氨酸 N 和酰胺羰基优先结合的结果。然而，在 CyP-cis 转化为 CyP-TS 时，N 末端部分 (Suc-Ala) 在 TS 中重新定位，两个氢键消失，一个氢键出现，另外两个氢键变弱。在此转化过程中，酶和肽之间的总疏水接触得以保留。因此，GS 和 TS 与酶的相互作用能量总体上非常相似。这不支持在顺反反应中通过K-m/K-Ts = 10(6) TS比GS结合更紧密的论点，当底物X残基从Gly < Trp < Ala < Leu改变时，肽的N末端部分在CyP-TS形成上的重新定位变得更加明显。我们认为，实验观察到的 DeltaS（不等于）和 DeltaH（不等于）值较大是由 N 端较大的转角造成的，这使得 DeltaG（不等于）变化很小。 Arg55 的位置和酰胺 C-N 键的 200 扭曲程度被认为是顺反异构化中近攻击构象 (NAC) 的标准。 NAC约占顺式异构体GS总数的30%。使用四种不同的底物观察到类似的 NAC 群体。这与酶活性对 X 残基的性质不敏感是一致的。此外，CyP.trans-AAPF 中的 NAC 群体与 CyP.cis-AAPF 中的 NAC 群体相当，这与 CyP 中顺反和反顺反应的类似实验测量速率一致。这些 NAC 在 CyP-cis 和 CyP.trans 中发现，仅类似于水反应中四种可能的 TS 配置之一。该 TS 结构（syn/exo）的身份与酶促反应中实验确定的 KIE 值一致。然而，活性位点的几何形状也与相同 KIE 测量在活性位点中未检测到的另一个 TS 结构（anti/exo）互补，这意味着 TS 的几何适合度不能成为酶促反应的单一决定因素。
The mechanism of cis-trans isomerization of prolyl peptides catalyzed by cyclophilin (CyP) was studied computationally via molecular dynamics (MD) simulations of the transition state (TS) and the cis and trans forms of the ground state (GS), when bound to CyP and when free in aqueous solution. The MD simulations include four enzyme-bound species of tetrapeptide (Suc-Ala-XC(=O)-NPro-Phe-pNA; X = Gly, Trp, Ala, and Leu). In water, the prolyl amide bond is favorably planar with the presence of conformers exhibiting :1,201 twist of the C-N dihedral. In the active site a hydrogen bond between the cis-prolyl amide carbonyl 0 and the backbone amide N-H of Asn102 retains the 201 twist of the C-N dihedral. The TS structure is characterized by a 900 twist of the amide C-N bond and a more favorable interaction with Asn102 due to the shorter distance between Asn102(HN) and the amide carbonyl O. The conformational change of cis - TS also involves pyramidalization of the amide N, which results in the formation of a hydrogen bond between the amide N and the guanidino group of Arg55. Both Asn102 and Arg55 are held in the same position in CyP.cis-isomer as in CyP.TS. In the ligand-free CyP the Arg55 guanidino group is highly disorganized and Asn102 is displaced 1 Angstrom from the position in the ligand-bound CyP. Thus, the organization of Arg55 and Asn102 occurs upon substrate binding. The geometrical complimentarity of the organized enzyme structure to the TS structure is a result of preferential binding of the proline N and the amide carbonyl of the TS compared to that of GS. However, the N-terminal part (Suc-Ala) becomes repositioned in the TS such that two hydrogen bonds disappear, one hydrogen bond appears and two other hydrogen bonds becomes weaker on the conversion of CyP-cis to CyP-TS. During this conversion, total hydrophobic contact between enzyme and the peptide is preserved. Thus, the interaction energies of GS and TS with enzyme are, as a whole, much alike. This does not support the contention that TS is bound more tightly than GS by K-m/K-Ts = 10(6) in the cis - trans reaction, Repositioning of the N-terminal part of the peptide on CyP-TS formation becomes more pronounced when the substrate X residue is changed from Gly < Trp < Ala < Leu. We propose that the larger turning of the N-terminus is responsible for the larger value of the experimentally observed DeltaS(not equal) and DeltaH(not equal), which sum up to little change in DeltaG(not equal). The positioning of the Arg55 and the degree of 200 twist of the amide C-N bond are considered as criteria for Near Attack Conformers (NACs) in cis-trans isomerization. NACs account for similar to30% of the total GS populations of the cis-isomer. Similar NAC populations were observed with four different substrates. This is consistent with the insensitivity of enzymatic activity to the nature of the X residue. Also, the NAC population in CyP.trans-AAPF was comparable to that in CyP.cis-AAPF, in accord with similar experimentally measured rates of the cis - trans and trans - cis reaction in CyP. These NACs, found in CyP-cis and CyP.trans, resemble only one of the four possible TS configurations in the water reaction. The identity of this TS structure (syn/exo) is in accord with experimentally determined KIE values in the enzymatic reaction.However, the geometry of the active site was also complementary to another TS structure (anti/exo) that was not detected in the active site by the same KIE measurements, implying that the geometrical fitness of the TS cannot be a single determining factor for enzymatic reactions.