Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate

Abstract

Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3), a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (K(D app)) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the K(m) for phenylbutyryl-CoA were 0.2 mM 34(-1)·sec(-1) and 5.3 μM compared to 4.0 mM(-1)·sec(-1) and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1

Schematic showing a proposed overall pathway for metabolism of phenylbutyrate to its final metabolite.

Figure 2

Monitoring the formation of the charge transfer complex with purified MCAD upon addition of increasing amounts of octanoyl-CoA. The absorbance maxima at ~370 nm and ~447 nm are reduced and a new peak centered at 570 nm appears with addition of increasing substrate. Selected scans are shown with octanoyl-CoA concentration at 0, 3.25, 7.1, 10.8, 15.6, 18.0, 21.5, and 28.2 μM. The inset shows the kinetics of these changes. Equation for the decrease at 447 nm is: y = −1 × 10−9x6 + 1 × 10−7x5 − 2 × 10−6x4 + 2 × 10−5x3 − 0.0003x2 − 0.008x + 0.3489. Equation for the increase at 570 nm is: y = 6 × 10−10x6 − 5 × 10−8x5 + 1 × 10−6x4 − 2 × 10−5x3 + 0.0003x2 + 0.0003x + 0.0008. Enzyme concentration was 25.2 μM.

Figure 3

Monitoring the formation of the charge transfer complex with purified MCAD upon addition of increasing amounts of phenylbutyryl-CoA. The absorbance maxima at ~370 nm and ~447 nm are reduced and a new peak centered at ~570 nm appears with addition of increasing substrate. Selected scans are shown with phenylbutyryl-CoA concentration at 0, 4.2, 8.3, 16.3, 24.1, 31.6, 40.7, and 80.2 μM. The inset shows the kinetics of these changes. Equation for the decrease at 447 nm is: y = 5 × 10−10x5 − 1 × 10−7x4 + 9 × 10−6x3 − 0.0002 x2 − 0.0061 x + 0.3707. Equation for the increase at 570 nm is: y = − 1 × 10−10x5 + 3 × 10−8x4 − 3 × 10−6x3 + 7 × 10−5x2 + 0.0012 x + 0.0036. Enzyme concentration was 25.2 μM.

Figure 4

Detailed proposed pathway of metabolism of phenylbutyrate to its active form, phenylacetyl-CoA.

Figure 5

Stick representation of MCAD active site residues and ligands with phenylbutyryl-CoA modeled in place of octanoyl-CoA. The crystal structure of pig MCAD with bound octanoyl-CoA (PDB: 3MDE, [20]) was used to create the model using MOE modeling software. The E376 carboxylate is the active site catalytic base responsible for the substrate C2 proton abstraction to initiate catalysis.