Pellett J D, Sabaj K M, Stephens A W, Bell A F, Wu J, Tonge P J, Stankovich M T
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Kolthoff and Smith Halls, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2000 Nov 14;39(45):13982-92. doi: 10.1021/bi0006464.
2,4-Hexadienoyl-coenzyme A (HD-CoA) has been used to investigate the redox and ionization properties of medium-chain acyl-CoA dehydrogenase (MCAD) from pig kidney. HD-CoA is a thermodynamically stabilized product analogue that binds tightly to oxidized MCAD (K(dox) = 3.5 +/- 0.1 microM, pH 7.6) and elicits a redox potential shift that is 78% of that observed with the natural substrate/product couple [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715]. The midpoint potential of the MCAD.HD-CoA complex exhibits a pH dependence that is consistent with the redox-linked ionization of two key glutamic acids as well as the flavin adenine dinucleotide (FAD) cofactor. The estimated ionization constants for Glu376-COOH (pK(a,ox) approximately 9.3) and Glu99-COOH (pK(a,ox) approximately 7.4) in the oxidized MCAD.HD-CoA complex indicate that while binding of the C(6) analogue makes Glu376 a stronger catalytic base (pK(a,ox) approximately 6.5, free MCAD), it has little effect on the pK of Glu99 (pK(a,ox) approximately 7.5, free MCAD) [Mancini-Samuelson, G. J., Kieweg, V., Sabaj, K. M., Ghisla, S., and Stankovich, M. T. (1998) Biochemistry 37, 14605-14612]. This finding is in agreement with the apparent pK of 9.2 determined for Glu376 in the human MCAD.4-thia-octenoyl-CoA complex [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. The pK(a)s estimated for Glu376 and Glu99 in the reduced pig kidney MCAD.HD-CoA complex, 9.8 and 8.6, respectively, suggest that both of these residues remain protonated in the charge-transfer complex under physiological conditions. Polarization of HD-CoA in the enzyme active site may contribute to the observed pK(a) and redox potential shifts. Consequently, the electronic structures of the product analogue in its free and MCAD-bound forms have been characterized by Raman difference spectroscopy. Binding to either the oxidized or reduced enzyme results in localized pi-electron polarization of the hexadienoyl C(1)=O and C(2)=C(3) bonds. The C(4)=C(5) bond, in contrast, is relatively unaffected by binding. These results suggest that, upon binding to MCAD, HD-CoA is selectively polarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of the oxidation state of the enzyme.
2,4-己二烯酰辅酶A（HD-CoA）已被用于研究猪肾中链酰基辅酶A脱氢酶（MCAD）的氧化还原和电离性质。HD-CoA是一种热力学稳定的产物类似物，它与氧化型MCAD紧密结合（K(dox)=3.5±0.1微摩尔，pH 7.6），并引起氧化还原电位的变化，该变化是天然底物/产物对所观察到变化的78%[伦恩，N.D.，斯坦科维奇，M.T.，和刘，H.（1990）《生物化学》29，3709 - 3715]。MCAD.HD-CoA复合物的中点电位表现出pH依赖性，这与两个关键谷氨酸以及黄素腺嘌呤二核苷酸（FAD）辅因子的氧化还原相关电离一致。氧化型MCAD.HD-CoA复合物中Glu376 - COOH（pK(a,ox)约为9.3）和Glu99 - COOH（pK(a,ox)约为7.4）的估计电离常数表明，虽然C(6)类似物的结合使Glu376成为更强的催化碱（pK(a,ox)约为6.5，游离MCAD），但对Glu99的pK影响很小（pK(a,ox)约为7.5，游离MCAD）[曼奇尼 - 萨缪尔森，G.J.，基韦格，V.，萨巴伊，K.M.，吉斯拉，S.，和斯坦科维奇，M.T.（1998）《生物化学》37，14605 - 14612]。这一发现与在人MCAD.4 - 硫代 - 辛烯酰辅酶A复合物中测定的Glu376的表观pK 9.2一致[鲁迪克，I.，吉斯拉，S.，和索普，C.（（1998）《生物化学》37，8437 - 8445]。在还原型猪肾MCAD.HD-CoA复合物中，Glu376和Glu99的估计pK(a)分别为9.8和8.6，这表明在生理条件下，这两个残基在电荷转移复合物中均保持质子化状态。HD-CoA在酶活性位点的极化可能导致观察到的pK(a)和氧化还原电位变化。因此，已通过拉曼差光谱对产物类似物的游离形式及其与MCAD结合形式的电子结构进行了表征。与氧化型或还原型酶的结合都会导致己二烯酰C(1)=O和C(2)=C(3)键的局部π电子极化。相比之下，C(4)=C(5)键受结合的影响相对较小。这些结果表明，与MCAD结合后，HD-CoA被选择性极化，使得配体的C(3)-H区域产生部分正电荷，而与酶的氧化态无关。
