Influence of two substrate analogs on thermodynamic properties of medium-chain acyl-CoA dehydrogenase

Abstract

The objective of this work was to identify the key structural functionalities of substrate or product that modulate the thermodynamic properties of medium-chain acyl-CoA dehydrogenase (MCAD). In order to achieve this, two classes of substrate analogues, acetyl-CoA and thioether-CoAs, were complexed with MCAD and their effects on the redox properties of MCAD were measured. A pH dependence study of the redox potential of uncomplexed MCAD allowed us to compare redox properties between complexed and uncomplexed MCAD and to calculate the dissociation constants of the analogues to the three redox states of MCAD. The results from this work indicate that these analogues are not influencing the thermodynamic behavior of MCAD in the same way as natural substrate. Thus, we propose that the following two key structural features of the binding ligand are necessary for mimicking the thermodynamic effects natural substrate has on MCAD: a thioester carbonyl on carbon 1 and a fatty acyl-CoA chain length around 8 carbon units. Furthermore, with the advent of structural knowledge, insights into the interactions of these structural features with MCAD and their influence on MCAD's highly regulated dehydrogenation mechanism are discussed.

Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues

“…From this finding, it can be concluded that the redox potential, E 1 , of the E-Fl ox /E-FlH • couple is more negative than E 2 , the E-FlH • /E-Fl red H -couple. The values for E 1 and E 2 cannot be estimated because of the small amount of semiquinone thermodynamically stabilized (<5%), which is substantially less than the 20% found for pig kidney MCADH under similar conditions (24). The redox potential determined for hwtMCADH (-0.114 V) is an average of E 1 and E 2 , and is thus best described as a midpoint potential.…”

“…For the purpose of comparison, the E m vs pH dependence of pig MCADH obtained at 25°C (24) has been reanalyzed using the same equation as with hwtMCADH. The results indicate some marked differences.…”

Protein Dynamics Enhance Electronic Coupling in Electron Transfer Complexes

“…Assuming that there is only a small change in redox potential of the individual flavins on formation of the ETF⅐MCAD complex, ⌬G for electron transfer from hydroquinone MCAD to oxidized ETF (Ϫ0.12 eV) and from semiquinone MCAD to oxidized ETF (Ϫ0.16 eV) was calculated from the known potentials of the semiquinone/hydroquinone (Ϫ94 mV) and oxidized/semiquinone (Ϫ134 mV) couples of MCAD (at pH ϭ 7.1 (36)) and the potential of the oxidized/semiquinone couple (ϩ22 mV) of human ETF (37). The reorganization energy, , was set to 0.7 eV.…”

Synthesis of Substrate Analogs for Glutaryl-CoA Dehydrogenase: 3-Thia-Glutaryl-CoA and 4-Nitrobutyryl-CoA

“…This behavior has been exhibited by other acyl-CoA dehydrogenases such as the short and medium chain acyl-CoA dehydrogenases (SCAD and MCAD). Through spectroelectrochemical studies of SCAD and MCAD, where each were complexed with different substrate, product, and substrate and product analog compounds, it was revealed that the electron transfer behavior of these enzymes is regulated by the binding of certain CoA compounds in the active site (2,3,4,5). The particular substrate analog syntheses reported here were designed to supply analogs that should bind to the GCD active site, enabling GCD spectroelectrochemical studies similar in design to those for SCAD and MCAD to be performed.…”