13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein.
13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein.
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胆色素原合酶的 13C NMR 研究：观察与 280,000 道尔顿蛋白质结合的中间体。
DOI：
10.1021/bi00388a012
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Markham,GD
中科院分区：
文献类型：
--
作者：
Jaffe,EK;Markham,GD
艾琳·K Jaffe*· 1和乔治D. Markham 8宾夕法尼亚大学牙科医学院生物化学系，宾夕法尼亚州费城19104-6002，和癌症研究所，福克斯蔡斯癌症中心，宾夕法尼亚州费城19111接收于1986年12月8日;修订版Mandarin pt接收于1987年3月11日摘要：用~（13）C NMR研究了[4-~（13）C]-5-氨基乙酰丙酸（[4-~（13）C] ALA）与胆色素原（PBG）合酶（5-氨基乙酰丙酸酯酶）（一种分子量为280000-道尔顿的蛋白质）的平衡复合物。[4- 13 C] ALA（化学位移= 205.9 ppm）形成[3，5- 13 C] PBG（化学位移= 121.0和123.0 ppm）。使用由[4- 13 C] ALA和[15 N] ALA的混合物制备的PBG分别将121.0和123.0 ppm共振归属于C5和C3。对于全酶和[4-I3 C] ALA形成的酶结合平衡复合物，观察到化学位移为121.5和127.2 ppm的两个等面积峰（线宽约50 Hz），表明主要物种可能是PBG的畸变形式。当存在过量的游离PBG时，它与结合的PBG缓慢交换，表明交换速率< 10 s-1，这与酶的周转速率一致。对于[4- 13 C] ALA和甲硫基磺酸甲酯（MMTS）修饰的PBG合成酶形成的复合物，其不催化PBG的形成，主要物种是席夫碱加合物（化学位移= 166.5 ppm，线宽~ 50 Hz）。游离ALA与席夫碱的交换缓慢。活化的MMT-修饰的酶-席夫碱配合物与I13镉和2-巯基乙醇的结果在损失的席夫碱信号和外观的结合PBG与相同的化学位移的结合平衡复合物与Zn（II）酶。没有观察到113 Cd-13 C耦合的分裂和增宽。核磁共振（NMR）1作为一种潜在的酶催化反应活性中心化学的探针，已被广泛应用。自从Mildred Cohn和同事首次使用31 P NMR研究与酶精氨酸激酶结合的底物之间的平衡络合物以来，已经过去了10多年（Rao等人，1976年）。虽然碳化学远比磷化学对酶催化反应的不适用性大，但13 C NMR作为酶活性位点的探针却受到1.1%的天然13 C同位素丰度的影响。低天然丰度指示同位素富集的底物分子的合成，而高天然丰度贡献来自蛋白质的碳的实质性共振强度（背景）。Nev-ertheless，麦肯齐等（1984）在最近的综述中预测，利用13 C NMR研究低于50 000道尔顿的酶-底物复合物，利用低温溶剂延长酶结合中间体的寿命，具有很好的前景。胆色素原（PBG）合酶（aka-氨基乙酰丙酸酯酶，EC 4.2. 1.24）催化不对称缩合，
Eileen K. Jaffe*· 1 and George D. Markham8 Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania 19104-6002, and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 Received December 8, 1986; Revised Manuscript Received March 11, 1987 abstract: 13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C] ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280 000-dalton protein.[4-13C] ALA (chemical shift= 205.9 ppm) forms [3, 5-13C] PBG (chemical shifts= 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C] ALA and [15N] ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-I3C] ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths~ 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of< 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formedfrom [4-13C] ALA and methyl methanethiosulfonate (MMTS) modified PBGsynthase, which does notcatalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift= 166.5 ppm, line width~ 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with I13Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn (II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.^ Nuclear magnetic resonance(NMR) 1 has long been rec-ognized as a potential probe of the active site chemistry of enzyme-catalyzed reactions. More than 10 years have passed since Mildred Cohn and co-workers first used 31P NMR to investigate the equilibrium complexbetween substrates bound to an enzyme, arginine kinase (Rao et al., 1976). Although carbon chemistry far exceeds phosphorus chemistry inap-plicability to enzyme-catalyzed reactions, 13C NMR as a probe of enzyme active sites suffers from the natural 1.1% isotopic abundance of 13C. The low natural abundance dictates synthesis of isotopically enriched substrate molecules, while the high natural abundance contributes substantial resonance intensity (background) from the carbons of the protein. Nev-ertheless, Mackenzie et al.(1984) in a recent review predict a promising future for the use of 13C NMR to study en-zyme-substrate complexes below 50 000daltons, using cryo-solvents to prolong the lifetime of enzyme-bound intermediates. Porphobilinogen (PBG) synthase (aka-aminolevulinate dehydratase, EC 4.2. 1.24) catalyzes the asymmetric conden-