Medium-Chain Acyl-Coenzyme A Dehydrogenase Bound to a Product Analogue, Hexadienoyl-Coenzyme A: Effects on Reduction Potential, pKa, and Polarization
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- 作者：Jackson D. Pellett ; Kim Marie Sabaj ; Avery W. Stephens ; Alasdair F. Bell ; Jiaquan Wu ; Peter J. Tonge ; and Marian T. Stankovich
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：November 14, 2000
- 年：2000
- 卷：39
- 期：45
- 页码：13982 - 13992
- 全文大小：129K
- 年卷期：v.39,no.45(November 14, 2000)
- ISSN：1520-4995
文摘
2,4-Hexadienoyl-coenzyme A (HD-CoA) has been used to investigate the redox and ionizationproperties of medium-chain acyl-CoA dehydrogenase (MCAD) from pig kidney. HD-CoA is athermodynamically stabilized product analogue that binds tightly to oxidized MCAD (Kdox = 3.5 ± 0.1
M, 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]. Themidpoint potential of the MCAD·HD-CoA complex exhibits a pH dependence that is consistent with theredox-linked ionization of two key glutamic acids as well as the flavin adenine dinucleotide (FAD) cofactor.The estimated ionization constants for Glu376-COOH (pKa,ox 
9.3) and Glu99-COOH (pKa,ox 7.4) inthe oxidized MCAD·HD-CoA complex indicate that while binding of the C6 analogue makes Glu376 astronger catalytic base (pKa,ox 6.5, free MCAD), it has little effect on the pK of Glu99 (pKa,ox 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 determinedfor Glu376 in the human MCAD·4-thia-octenoyl-CoA complex [Rudik, I., Ghisla, S., and Thorpe, C.(1998) Biochemistry 37, 8437-8445]. The pKas estimated for Glu376 and Glu99 in the reduced pig kidneyMCAD·HD-CoA complex, 9.8 and 8.6, respectively, suggest that both of these residues remain protonatedin the charge-transfer complex under physiological conditions. Polarization of HD-CoA in the enzymeactive site may contribute to the observed pKa and redox potential shifts. Consequently, the electronicstructures of the product analogue in its free and MCAD-bound forms have been characterized by Ramandifference spectroscopy. Binding to either the oxidized or reduced enzyme results in localized 
-electronpolarization of the hexadienoyl C(1)=O and C(2)=C(3) bonds. The C(4)=C(5) bond, in contrast, isrelatively unaffected by binding. These results suggest that, upon binding to MCAD, HD-CoA is selectivelypolarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of theoxidation state of the enzyme.
M, 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]. Themidpoint potential of the MCAD·HD-CoA complex exhibits a pH dependence that is consistent with theredox-linked ionization of two key glutamic acids as well as the flavin adenine dinucleotide (FAD) cofactor.The estimated ionization constants for Glu376-COOH (pKa,ox 9.3) and Glu99-COOH (pKa,ox 7.4) inthe oxidized MCAD·HD-CoA complex indicate that while binding of the C6 analogue makes Glu376 astronger catalytic base (pKa,ox 6.5, free MCAD), it has little effect on the pK of Glu99 (pKa,ox 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 determinedfor Glu376 in the human MCAD·4-thia-octenoyl-CoA complex [Rudik, I., Ghisla, S., and Thorpe, C.(1998) Biochemistry 37, 8437-8445]. The pKas estimated for Glu376 and Glu99 in the reduced pig kidneyMCAD·HD-CoA complex, 9.8 and 8.6, respectively, suggest that both of these residues remain protonatedin the charge-transfer complex under physiological conditions. Polarization of HD-CoA in the enzymeactive site may contribute to the observed pKa and redox potential shifts. Consequently, the electronicstructures of the product analogue in its free and MCAD-bound forms have been characterized by Ramandifference spectroscopy. Binding to either the oxidized or reduced enzyme results in localized -electronpolarization of the hexadienoyl C(1)=O and C(2)=C(3) bonds. The C(4)=C(5) bond, in contrast, isrelatively unaffected by binding. These results suggest that, upon binding to MCAD, HD-CoA is selectivelypolarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of theoxidation state of the enzyme.