EVALUATION OF THE FACTORS INFLUENCING REACTIVITY AND STEREOSPECIFICITY IN NAD(P)H DEPENDENT DEHYDROGENASE ENZYMES
EVALUATION OF THE FACTORS INFLUENCING REACTIVITY AND STEREOSPECIFICITY IN NAD(P)H DEPENDENT DEHYDROGENASE ENZYMES
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DOI：
10.1021/ja00059a005
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发表时间：
1993-03-24
影响因子：
15
通讯作者：
BRUICE, TC
中科院分区：
文献类型：
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作者：
ALMARSSON, O;BRUICE, TC
计算了 N-(1,β-核糖基)-1,4-二氢烟酰胺 (3H) 和 N-(1,β-核糖基)烟酰胺 (3) 构象的 AM1 势能，由烟酰胺环变形角 α(C) 和 α(N) 以及扭转角 X(n) 和 X(am) 定义。 X(n)是烟酰胺环相对于核糖环的旋转角，构象异构体被指定为顺式（烟酰胺-CONH2和核糖环O靠近）和反式（烟酰胺-CONH2和核糖环O远离）。角度α(C)和α(N)反映了烟酰胺N和C4与C2、C3、C5和C6的平面外弯曲的程度，以提供准船和准半椅构象。当1,4-二氢烟酰胺环呈准船形时，核糖醚O上的反键轨道与烟酰胺环N上的非共享电子对的关系被描述为围平面或反围平面。然后，准船构象可被描述为反反周面（1a）、顺反周面（1b）、反周面（1c）和顺反周面（1d）。 1,4-二氢吡啶环变形为准船或准半椅构象（α(C) + α(N) 小于或等于 20 度且 α(C) 大于或等于 α(N)）所消耗的 AM1 势能小于通过转移羰基官能团还原过程中伴随的过渡态稳定所获得的增益。 C4 处的赝轴氢。当 alpha(C) = 15 度和 alpha(N) = 5 度时，作为 X(n) 函数的 3 势能相对于其全局最小值提高了平均值 16 kcal/mol。将3H的扁平1,4-二氢烟酰胺弯曲至α(C)=15度、α(N)=5度，需要花费1.8kcal/mol，而活化焓比扁平烟酰胺降低了6kcal/mol。在弯曲构象中，初始态{3H + H2C=O...HImH+}和直接产物态{3 + H3COH...ImH}势能差别不大。从能量上来说，H(R) 或 H(S) 是否是赝轴位置处的转移实体几乎没有区别。根据保存在 Brookhaven 数据库中的结构，对于 A 特异性脱氢酶，X(n) = -87 度到 -140 度（构象 1a），对于 B 特异性脱氢酶，X(n) = 45 度到 66 度（构象 1b）。未结合 (AM1) 和酶结合 (Brookhaven 数据库) 还原和氧化辅因子的 X(n) 首选值的不匹配对辅因子结合的平衡常数几乎没有影响，有利于还原形式。 CHARM(m) 分子动力学模拟显示，当 3H 未与酶结合时，3H 的 α(C) + α(N) 向 A 侧和 B 侧的变形是等能的。使用角鲨肌肉乳酸脱氢酶进行的相同实验表明，NAD+ 和 NADH 扭转角 X(n) 和 X(am) 以及 C4 的褶皱角 α(C) 的运动非常灵活，正如 AM1 计算所预测的那样。使用龙虾 D-甘油醛 3-磷酸脱氢酶、干酪乳杆菌二氢叶酸还原酶和猪心苹果酸脱氢酶进行的分子动力学模拟也表明 NADH 的 C4 的褶皱角 α(C) 非常灵活。对于各种脱氢酶，NADH 的褶皱运动是各向异性的，使得 C4 主要向底物结合位点弯曲。对于每种酶，辅因子远端面上的疏水残基造成的空间位阻是造成这种各向异性运动的原因。在角鲨乳酸脱氢酶丙酮酸-NADH复合物中，C4处的可转移假轴H(R)与底物的羰基碳处于范德华接触范围内。此外，动力学计算表明，HIS 193 的质子化咪唑基团不仅需要作为一般酸催化剂，而且还需要丙酮酸的正确排列。使用活性位点周围酶的 X 射线坐标进行 AM1 计算，并使用整个酶进行分子动力学计算，检查了角鲨乳酸脱氢酶的重要催化特征。这些特征如下：（i）在基态下，所有反应物接近范德华半径内； (ii)在过渡态，质子从HIS 193的咪唑鎓阳离子(ImH-2+)到底物羰基氧的转移几乎完成，而氢从1,4-二氢烟酰胺到羰基碳的转移大约在中途。 ImH-2+ 的质子转移的后期过渡态是由 ImH-2+ 的排斥性正电荷和 ARG 106 的精氨酸胍阳离子取代基促进的。据推测，催化的主要驱动力是基态的空间压缩、质子转移的延迟（在羰基碳上提供大量的部分正电荷）以及预平衡时起皱。 1,4-二氢烟酰胺环通过准轴氢的转移形成准船构象。 A 侧或 B 侧氢转移的有利性是通过屏蔽 1,4-二氢烟酰胺环的一侧来确定的，这可以防止底物进入未屏蔽侧并防止动态变形。早期提出的脱氢酶进化方式是通过让 NADH 在 H(R) 转移（A 侧）中呈现较弱的还原剂反周面构象 (1a) 和在 H(S) 转移（B 侧）中呈现较强的还原剂顺反周面构象 (1b) 来平衡其动态，但根据这些构象的 AM1 势能（即 1a 和 1b 应该是可比较的），这一提议是不正确的还原剂）。在干酪乳杆菌二氢叶酸还原酶中，THR 45、ILE 13 和 ALA 97 的氧功能与 NADPH 的 C2-H、C4-H 和 C6-H 的氢的弱极性相互作用并不代表先前认为在辅因子的激活中重要的氢键。一般而言，在脱氢酶中，向内指向烟酰胺环的氨基酸氧官能团参与脱辅基酶中与水分子的氢键，从而保持辅因子结合位点开放。
Calculations of AM1 potential energies for conformations of N-(1,beta-ribosyl)-1,4-dihydronicotinamide (3H) and N-(1,beta-ribosyl)nicotinamide (3), as defined by the nicotinamide ring deformation angles alpha(C) and alpha(N) and the torsion angles X(n) and X(am), have been carried out. X(n) is the angle of rotation of the nicotinamide ring relative to the ribose ring and the conformers are designated as syn (nicotinamide -CONH2 and ribose ring O are, close) and anti (nicotinamide -CONH2 and ribose ring O are distant). The angles alpha(C) and alpha(N) reflect the degree of bending of nicotinamide N and C4 out of the plane with C2, C3, C5, and C6 to provide quasi-boat and quasi-half-chair conformations. With the 1,4-dihydronicotinamide ring in a quasi-boat geometry, the relationship of the anti-bonding orbital on the ribose ether O and the unshared electron pair on the nicotinamide ring N is described as periplanar or antiperiplanar. The quasi-boat conformations may then be described as anti antiperiplanar (1a), syn antiperiplanar (1b), anti periplanar (1c), and syn periplanar (1d). The AM1 potential energies expended in the deformation of the 1,4-dihydropyridine ring to quasi-boat or quasi-half-chair conformations (with alpha(C) + alpha(N) less-than-or-equal-to 20-degrees and alpha(C) greater-than-or-equal-to alpha(N)) arc less than the gain in the accompanying stabilization of the transition state in the reduction of a carbonyl function by transfer of the pseudoaxial hydrogen at C4. With alpha(C) = 15-degrees and alpha(N) = 5-degrees, the potential energies of 3 as a function of X(n) is raised relative to its global minimum by an average value of 16 kcal/mol. To bend the flat 1,4-dihydronicotinamide of 3H such that alpha(C) = 15-degrees and alpha(N) = 5-degrees costs 1.8 kcal/mol, while the activation enthalpy is decreased by 6 kcal/mol compared with the flat nicotinamide. In the bent conformation, the initial state {3H + H2C=O...HImH+} and immediate product state {3 + H3COH...ImH} potential energies are not greatly different. Energetically, there is little difference as to whether H(R) or H(S) are the transferred entities at the pseudoaxial positions. From structures deposited in the Brookhaven database, X(n) = -87-degrees to -140-degrees (conformation 1a) for A-specific and X(n) = 45-degrees to 66-degrees (conformation 1b) for B-specific dehydrogenases. The mismatch in the preferred values of X(n) for unbound (AM1) and enzyme bound (Brookhaven database) reduced and oxidized cofactors has little influence on the equilitrium constants for cofactor binding which favors the reduced form. CHARM(m) molecular dynamics simulations show the deformation of the alpha(C) + alpha(N) of 3H to A- and B-sides is isoenergetic when 3H is not enzyme bound. The same experiments with dogfish muscle lactate dehydrogenase show the motions of the NAD+ and NADH torsional angles X(n) and X(am), as well as the puckering angle alpha(C) for C4, are quite flexible as predicted by AM1 calculations. Molecular dynamics simulations with lobster D-glyceraldehyde 3-phosphate dehydrogenase, L. casei dihydrofolate reductase, and porcine heart malate dehydrogenase also show the puckering angle alpha(C) for C4 of NADH to be quite flexible. With the various dehydrogenases the puckering motion of NADH is anisotropic, such that the C4 predominantly bends toward the substrate binding site.For each enzyme, steric hindrance by hydrophobic residues on the distal face of the cofactor are responsible for this anisotropic movement. In the dogfish lactate dehydrogenase pyruvate-NADH complex, the transferable pseudoaxial H(R) at C4 comes within van der Waals contact with the carbonyl carbon of the substrate. Further, dynamics calculations indicate that the protonated imidazole group of HIS 193 is required not only as a general-acid catalyst but also for the correct alignment of pyruvate. The important catalytic features of the dogfish lactate dehydrogenase were examined by AM1 calculations using the X-ray coordinates for the enzyme around the active site and by molecular dynamics calculations using the entire enzyme. These features are the following: (i) in the ground state, approach of all reactants to within van der Waals radii; (ii) in the transition state, proton transfer from imidazolium cation (ImH-2+) of HIS 193 to substrate carbonyl oxygen is almost complete while hydrogen transfer from 1,4-dihydronicotinamide to carbonyl carbon is about midway. The late transition state for proton transfer from ImH-2+ is facilitated by the repulsive positive charges of ImH-2+ and the arginine guanidinium cation substituent of ARG 106. It is proposed that the major driving forces in the catalysis are the steric compression in the ground state, the lateness of proton transfer which provides a large partial positive charge on the carbonyl carbon, and the preequilibrium puckering of the 1,4-dihydronicotinamide ring to quasi-boat conformation with transfer of the pseudoaxial hydrogen. The favoring of A- or B-side hydrogen transfer is determined by shielding of one side of the 1,4-dihydronicotinamide ring, which prevents access of substrate to and dynamic deformation at the unblocked side. An earlier proposal that dehydrogenases evolved in such a manner to level their dynamics by having NADH assume the weaker reductant anti antiperiplanar conformation (1a) in H(R) transfer (A-side) and the stronger reductant syn antiperiplanar conformation (1b) in H(S) transfer (B-side) cannot be correct on the basis of the AM1 potential energies of these conformations (i.e., 1a and 1b should be comparable reducing agents). In L. casei dihydrofolate reductase, the weakly polar interactions of oxygen functions of THR 45, ILE 13, and ALA 97 with the hydrogens of C2-H, C4-H, and C6-H of NADPH do not represent hydrogen bonds previously thought to be important in the activation of the cofactor. In the dehydrogenases in general, the amino acid oxygen functionalities which point inwards toward the nicotinamide ring are involved in hydrogen bonds to water molecules in the apoenzyme thereby keeping the cofactor binding site open.