Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica:: A comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase
Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica:: A comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase
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DOI：
10.1021/bi972115u
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发表时间：
1998-03-03
期刊：
影响因子：
2.9
通讯作者：
Duine, JA
中科院分区：
文献类型：
--
作者：
Piersma, SR;Visser, AJWG;Duine, JA
甲醇拟无枝酸菌（Amycolatopsis methanolica）的烟蛋白（含NADH）醇脱氢酶的NADH吸收光谱在326 nm处具有最大值。还原的酶结合吡啶二核苷酸可被乙醛可逆氧化。与酶结合的NADH的荧光激发光谱在325 nm处具有最大值。在290 nm处激发时，从色氨酸到酶结合的NADH的能量转移可以忽略不计。与酶结合的NADH的荧光发射光谱（在325 nm处激发）在422 nm处具有最大值。的荧光强度增强了3倍后，结合异丁酰胺（Kd = 59 μ M）。异丁酰胺作为竞争性抑制剂（Ki = 46 μ M）相对于电子受体NDMA（N，N-二甲基-p-亚硝基苯胺），它结合到酶含有还原辅因子。非反应性底物类似物三氟乙醇作为相对于底物乙醇（Ki = 1.6 μ M）的竞争性抑制剂，其与含有氧化辅因子的酶结合。含有NADH的酶和含有NAD（+）的酶的远紫外圆二色性光谱是相同的，表明在辅因子的氧化或还原后没有发生主要的构象变化。与酶结合的NADH的近紫外圆二色性光谱在323 nm处具有最小值（Δ λ = -8.6 M-1 cm（-1））。酶结合的NADH的荧光各向异性衰减表明没有旋转自由的NADH辅因子。这意味着刚性环境以及缺乏荧光团的运动。与酶结合的NADH的平均荧光寿命在20 ℃下为0.29 ns，并且可以被分解成至少三个组分（在0.13-0.96 ns的范围内）。当异丁酰胺与含酶的NADH结合时，平均激发态寿命增加到1.02 ns，并可分解为两个组分（0.37和1.11 ns）。与其他NADH-脱氢酶复合物相比，与烟蛋白醇脱氢酶结合的NADH的光谱具有蓝移最大值，但与马肝醇脱氢酶结合的NADH观察到的光谱相当。与酶结合的NADH复合物相比，与烟蛋白结合的NADH的荧光寿命非常短，也与马肝醇脱氢酶结合的NADH相比。在烟蛋白醇脱氢酶活性位点的辅因子-蛋白质相互作用比马肝醇脱氢酶更刚性和非极性。结合到烟蛋白醇脱氢酶的NADH的光学性质与结合到来自大肠杆菌的UDP-半乳糖差向异构酶的NADH（紧密地）的光学性质有很大不同。这表明，尽管浴酶具有NAD（H）作为不可交换的辅因子，但NADH结合位点是完全不同的。
The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alcohol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm. Reduced enzyme-bound pyridine dinucleotide could be reversibly oxidized by acetaldehyde. The fluorescence excitation spectrum for NADH bound to the enzyme has a maximum at 325 nm. Upon excitation at 290 nm, energy transfer from tryptophan to enzyme-bound NADH was negligible. The fluorescence emission spectrum (excitation at 325 nm) for NADH bound to the enzyme has a maximum at 422 nm. The fluorescence intensity is enhanced by a factor of 3 upon binding of isobutyramide (K-d = 59 mu M). Isobutyramide acts as competitive inhibitor (Ki = 46 mu M) with respect to the electron acceptor NDMA (N,N-dimethyl-p-nitrosoaniline), which binds to the enzyme containing the reduced cofactor. The nonreactive substrate analogue trifluoroethanol acts as a competitive inhibitor with respect to the substrate ethanol (K-i = 1.6 mu M), which binds to the enzyme containing the oxidized cofactor. Far-UV circular dichroism spectra of the enzyme containing NADH and the enzyme containing NAD(+) were identical, indicating that no major conformational changes occur upon oxidation or reduction of the cofactor. Near-UV circular dichroism spectra of NADH bound to the enzyme have a minimum at 323 nm (Delta epsilon = -8.6 M-1 cm(-1)). The fluorescence anisotropy decay of enzyme-bound NADH showed no rotational freedom of the NADH cofactor. This implies a rigid environment as well as lack of motion of the fluorophore. The average fluorescence lifetime of NADH bound to the enzyme is 0.29 ns at 20 degrees C and could be resolved into at least three components (in the range 0.13-0.96 ns). Upon binding of isobutyramide to the enzyme-containing NADH, the average excited-state Lifetime increased to 1.02 ns and could be resolved into two components (0.37 and 1.11 ns). The optical spectra of NADH bound to nicotinoprotein alcohol dehydrogenase have blue-shifted maxima compared to other NADH-dehydrogenase complexes, but comparable to that observed for NADH bound to horse liver alcohol dehydrogenase. The fluorescence lifetime of NADH bound to the nicotinoprotein is very short compared to enzyme-bound NADH complexes, also compared to NADH bound to horse Liver alcohol dehydrogenase. The cofactor-protein interaction in the nicotinoprotein alcohol dehydrogenase active site is more rigid and apolar than that in horse liver alcohol dehydrogenase. The optical properties of NADH bound to nicotinoprotein alcohol dehydrogenase differ considerably from NADH (tightly) bound to UDP-galactose epimerase from Escherichia coli. This indicates that although bath enzymes have NAD(H) as nonexchangeable cofactor, the NADH binding sites are quite different.