Engst S, Vock P, Wang M, Kim J J, Ghisla S
Faculty of Biology, University of Konstanz, Germany.
Biochemistry. 1999 Jan 5;38(1):257-67. doi: 10.1021/bi9815041.
The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acyl-coenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3'-deoxy-FAD and ribityl-2'-deoxy-FAD. 3'-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl side-chain 3'-OH group does not play any particular role in cofactor binding or catalysis. 2'-Deoxy-FAD-MCADH was characterized using the natural substrate C8CoA as well as various substrate and transition-state analogues. Substrate dehydrogenation in 2'-deoxy-FAD-MCADH is approximately 1.5 x 10(7)-fold slower than that of native MCADH, indicating that disruption of the hydrogen bond between 2'-OH and substrate thioester carbonyl leads to a substantial transition-state destabilization equivalent to approximately 38 kJ mol-1. The alphaC-H microscopic pKa of the substrate analogue 3S-C8CoA, which undergoes alpha-deprotonation on binding to MCADH, is lowered from approximately 16 in the free state to approximately 11 (+/-0.5) when bound to 2'-deoxy-FAD-MCADH. This compares with a decrease of the same pKa to approximately 5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of approximately 11 pK units, i.e., approximately 65 kJ mol-1 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of approximately 6 pK units ( approximately 35 kJ mol-1) between MCADH and 2'-deoxy-FAD-MCADH is taken as the level of stabilization of the substrate carbanionic species caused by the interaction with the FAD-2'-OH. This energetic parameter derived from the kinetic experiments (stabilization of transition state) is in agreement with those obtained from static experiments (lowering of alphaC-H microscopic pKa of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the two single H-bonds involved in substrate activation (Glu376amide-N-H and ribityl-2'-OH) thus appear to behave additively toward the total effect. The crystal structures of native pMCADH and of 2'-deoxy-FAD-MCADH complexed with octanoyl-CoA/octenoyl-CoA show unambiguously that the FAD cofactor and the substrate/product bind in an identical fashion, implying that the observed effects are mainly due to (the absence of) the FAD-ribityl-2'-OH hydrogen bond. The large energy associated with the 2'-OH hydrogen bond interaction is interpreted as resulting from the changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct involvement of a flavin cofactor side chain in catalysis.
猪肾中链特异性酰基辅酶A（CoA）脱氢酶（MCADH）的黄素腺嘌呤二核苷酸（FAD）辅因子已被核糖基-3'-脱氧-FAD和核糖基-2'-脱氧-FAD取代。3'-脱氧-FAD-MCADH的性质与天然MCADH非常相似，这表明FAD-核糖基侧链的3'-OH基团在辅因子结合或催化中不发挥任何特殊作用。使用天然底物C8CoA以及各种底物和过渡态类似物对2'-脱氧-FAD-MCADH进行了表征。2'-脱氧-FAD-MCADH中的底物脱氢速度比天然MCADH慢约1.5×10⁷倍，这表明2'-OH与底物硫酯羰基之间氢键的破坏导致了相当于约38 kJ·mol⁻¹的显著过渡态失稳。底物类似物3S-C8CoA在与MCADH结合时会发生α-去质子化，其αC-H微观pKa从游离状态下的约16降低到与2'-脱氧-FAD-MCADH结合时的约11（±0.5）。与之相比，在与未修饰的hwtMCADH形成的复合物中，相同的pKa降低到约5，这对应于约11 pK单位的pK位移，即约65 kJ·mol⁻¹[Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848 - 1860]。MCADH和2'-脱氧-FAD-MCADH之间这种约6 pK单位（约35 kJ·mol⁻¹）的效应差异被视为与FAD-2'-OH相互作用导致的底物碳负离子物种的稳定水平。从动力学实验得出的这个能量参数（过渡态的稳定）与从静态实验获得的参数一致（类似物αC-H微观pKa的降低，即阴离子过渡态类似物的稳定）。因此，参与底物活化的两个单氢键（Glu376酰胺-N-H和核糖基-2'-OH）的贡献似乎对总效应具有加和性。天然pMCADH以及与辛酰-CoA/辛烯酰-CoA复合的2'-脱氧-FAD-MCADH的晶体结构明确表明，FAD辅因子和底物/产物以相同方式结合，这意味着观察到的效应主要是由于（缺少）FAD-核糖基-2'-OH氢键。与2'-OH氢键相互作用相关的大量能量被解释为是由亲脂性底物结合引起的电荷变化和疏水性增加所致。这是第一个证明黄素辅因子侧链直接参与催化的例子。
