Mancini-Samuelson G J, Kieweg V, Sabaj K M, Ghisla S, Stankovich M T
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Biochemistry. 1998 Oct 13;37(41):14605-12. doi: 10.1021/bi981414w.
The modulation of the electron-transfer properties of human medium-chain acyl-CoA dehydrogenase (hwtMCADH) has been studied using wild-type and site-directed mutants by determining their midpoint potentials at various pH values and estimating the involved pKs. The mutants used were E376D, in which the negative charge is retained; E376Q, in which one negative charge (pKa approximately 6. 0) is removed from the active center; E99G, in which a different negative charge (pKa approximately 7.3) also is affected; and E376H (pKa approximately 9.3) in which a positive charge is present. Em for hwtMCADH at pH 7.6 is -0.114 V. Results for the site-directed mutants indicate that loss of a negative charge in the active site causes a +0.033 V potential shift. This is consistent with the assumption that electrostatic interactions (as in the case of flavodoxins) and specific charges are important in the modulation of the electron-transfer properties of this class of dehydrogenases. Specifically, these charge interactions appear to correlate with the positive Em shift observed upon binding of substrate/product couple to MCADH [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715], which coincides with a pK increase of Glu376-COOH from approximately 6 to 8-9 [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. From the pH dependence of the midpoint potentials of hwtMCADH two mechanistically important ionizations are estimated. The pKa value of approximately 6.0 is assigned to the catalytic base, Glu376-COOH, in the oxidized enzyme based on comparison with the pH behavior of the E376H mutant, it thus coincides with the pK value recently estimated [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The pKa of approximately 7.1 is assigned to Glu376-COOH in reduced hwtMCADH. Comparable values for these pKas for Glu376-COOH in pig kidney MCADH are pKox = 6.5 and pKred = 7.9. The Em measured for K304E-MCADH (a major mutant resulting in a deficiency syndrome) is essentially identical to that of hwtMCADH, indicating that the disordered enzyme has an intact active site.
通过测定野生型和定点突变体在不同pH值下的中点电位并估算相关的pK值，研究了人类中链酰基辅酶A脱氢酶（hwtMCADH）电子转移特性的调节。所使用的突变体包括：E376D，其保留了负电荷；E376Q，其从活性中心去除了一个负电荷（pKa约为6.0）；E99G，其另一个不同的负电荷（pKa约为7.3）也受到影响；以及E376H（pKa约为9.3），其存在一个正电荷。hwtMCADH在pH 7.6时的Em为-0.114 V。定点突变体的结果表明，活性位点中负电荷的丧失导致电位正移+0.033 V。这与以下假设一致，即静电相互作用（如黄素氧还蛋白的情况）和特定电荷在这类脱氢酶电子转移特性的调节中很重要。具体而言，这些电荷相互作用似乎与底物/产物偶联物与MCADH结合时观察到的正Em位移相关[Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709 - 3715]，这与Glu376 - COOH的pK从约6增加到8 - 9相吻合[Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437 - 8445]。根据hwtMCADH中点电位的pH依赖性，估算了两个具有重要机制意义的电离。基于与E376H突变体pH行为的比较，将约6.0的pKa值赋予氧化酶中的催化碱基Glu376 - COOH，因此它与最近估算的pK值一致[Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848 - 1860]。将约7.1的pKa赋予还原型hwtMCADH中的Glu376 - COOH。猪肾MCADH中Glu376 - COOH的这些pKa的可比数值为pKox = 6.5和pKred = 7.9。对K304E - MCADH（一种导致缺陷综合征的主要突变体）测得的Em与hwtMCADH的基本相同，表明该无序酶具有完整的活性位点。
