Redox properties of human medium-chain acyl-CoA dehydrogenase, modulation by charged active-site amino acid residues.
Redox properties of human medium-chain acyl-CoA dehydrogenase, modulation by charged active-site amino acid residues.
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人中链酰基辅酶A脱氢酶的氧化还原特性，通过带电荷的活性位点氨基酸残基进行调节。
DOI：
10.1021/bi981414w
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Stankovich,MT
中科院分区：
文献类型：
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作者：
Mancini-Samuelson,GJ;Kieweg,V;Sabaj,KM;Ghisla,S;Stankovich,MT
通过测定不同 pH 值下的中点电位并估计相关 pK，使用野生型和定点突变体研究了人中链酰基辅酶 A 脱氢酶 (hwtMCADH) 电子转移特性的调节。使用的突变体是E376D，其中保留了负电荷； E376Q，其中1个负电荷（pKa≈6.0）从活性中心被去除； E99G，其中不同的负电荷（pKa≈7.3）也受到影响；和 E376H (pKa≈ 9.3)，其中存在正电荷。 hwtMCADH 在 pH 7.6 时的 Em 值为 -0.114 V。定点突变体的结果表明，活性位点中负电荷的丢失会导致 +0.033 V 电位变化。这与静电相互作用（如黄素氧还蛋白的情况）和特定电荷对于调节此类脱氢酶的电子转移特性很重要的假设是一致的。具体来说，这些电荷相互作用似乎与底物/产物对与 MCADH 结合时观察到的正 Emshift 相关 [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990)Biochemistry29, 3709−3715]，这与 Glu376-COOH 的 pKincrease 从 ∼6 增加到 8−9 [Rudik, I., Ghisla, S. 和 Thorpe, C. (1998) 生物化学 37, 8437−8445]。根据 hwtMCADH 中点电势的 pH 依赖性，估计了两个机械上重要的电离。根据与 E376H 突变体的 pH 行为的比较，氧化酶中的催化碱基 Glu376-COOH 的 pKa 值约为 6.0，因此它与最近估计的 pK 值一致 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998)Biochemistry 37, 1848−1860]。 pKaof ∼7.1 被指定为还原 hwtMCADH 中的 Glu376-COOH。猪肾 MCADH 中 Glu376-COOH 的这些 pKa 的可比值为 pKox= 6.5 和 pKred= 7.9。 K304E-MCADH（一种导致缺陷综合征的主要突变体）的测量值与 hwtMCADH 的测量值基本相同，表明紊乱的酶具有完整的活性位点。
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≈ 6.0) is removed from the active center; E99G, in which a different negative charge (pKa≈ 7.3) also is affected; and E376H (pKa≈ 9.3) in which a positive charge is present.Emfor 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 positiveEmshift observed upon binding of substrate/product couple to MCADH [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990)Biochemistry29, 3709−3715], which coincides with a pKincrease of Glu376-COOH from ∼6 to 8−9 [Rudik, I., Ghisla, S., and Thorpe, C. (1998)Biochemistry37, 8437−8445]. From the pH dependence of the midpoint potentials of hwtMCADH two mechanistically important ionizations are estimated. The pKavalue of ∼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 pKvalue recently estimated [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998)Biochemistry 37, 1848−1860]. The pKaof ∼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. TheEmmeasured 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.