Medium-chain acyl-coenzyme A dehydrogenase bound to a product analogue, hexadienoyl-coenzyme A: effects on reduction potential, pK(a), and polarization.
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.
Pellett
J D
JD
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Kolthoff and Smith Halls, Minneapolis, Minnesota 55455, USA.
Sabaj
K M
KM
Stephens
A W
AW
Bell
A F
AF
Wu
J
J
Tonge
P J
PJ
Stankovich
M T
MT
D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004563:Electrochemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011199:Potentiometry; D013059:Spectrum Analysis, Raman; D013552:Swine
D000214:Acyl Coenzyme A; D046911:Macromolecular Substances; C417225:hexadienoyl-coenzyme A; D005182:Flavin-Adenine Dinucleotide; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase