Biochemical Studies on Recombinant Human Isobutyryl-CoA Dehydrogenase

Ibrahim, Ibrahim

doi:10.3844/ajbbsp.2011.84.89

Cited by 4 publications

(11 citation statements)

References 24 publications

(23 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The isobutyryl-CoA binding constant values are varying strongly with the pH, as their logarithmic values decrease with increasing the pH with better binding at pH 7.5-9. On the other hand, the Cys residues have role(s) in the reactivity of the IBD as mentioned in our previous work [20] . To further study the role of these Cys residues we study the protonation states of the Cys residues which they depend on complex electrostatic interactions between Cys residues themselves and with the surrounding environment including neighbor residues and the pH of the buffer.…”

Section: Fig (1)mentioning

confidence: 63%

“…Methods. The enzyme was purified to homogeneity using the method described before [20] . Stopped-flow experiments were carried out using Stopped-flow spectrophotometer where the fastest acquisition time of 1 spectrum/0.7 ms in the range of 300-650 nm.…”

Section: Methodsmentioning

confidence: 99%

“…The concentrations of saturated acyl-CoAs and FAD were calculated according to their extinction coefficient ( 260 ) values at 260 nm, 15.4 mM -1 cm -1 [23] and 10.8 mM -1 cm -1 [24] , respectively. All anaerobic reactions and pH dependent activities were carried out as described in [20] . Protonation of the Cys residues at different pHs were simulated using H++ atomistic molecular modeling and simulations web server (http:// biophysics.cs.vt.edu/) using the PDB files 1rx0.pdb and 2JIF.pdb for human isobutyryl-CoA and short branched-chain acyl-CoA dehydrogenases, respectively, downloaded from Research Collaboratory for Structural Bioinformatics-Protein Data Bank database (RCSB PDB) (http://www.rcsb.org) [25,26] .…”

Section: Methodsmentioning

confidence: 99%

“…In comparison with IVD, the lateral expansion of the binding cavity in IVD is not observed in IBD [19] . IBD has many unique features including its high substrate specificity to isobutyryl-CoA and high Cysteines content (13 residues) which were shown to be important for its activity [20] . Here we study the IBD substrate, isobutyryl-CoA, binding and reduction by using the Stopped-flow kinetic analysis.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Stopped-flow kinetics and cysteine residue protonation of human isobutyryl-CoA dehydrogenase

2017

Egyptian Journal of Pure and Applied Science

View full text Add to dashboard Cite

Human Isobutyryl-CoA Dehydrogenase, IBD, is involved in the valine amino acid catabolism. It catalyzes with a high specificity the reduction of the isobutyryl-CoA to methylacrylyl-CoA in contrary to the rat Short-Branched Chain Acyl-CoA Dehydrogenase, SBCAD, which is able to utilize both 2methylbutyryl-CoA and isobutyryl-CoA that emerged from Ile and Val degradation, respectively. Here, the IBD stopped-flow kinetic was studied and it was found that IBD reduction reaction has two phases, the faster one was exponential at pH 9.0 while it was much slower and multi exponential in pH 6.5. Also, it was found that isobutyryl-CoA binds IBD in two steps, where the first step was the faster one. The protonation of the high IBD Cys content was computed by the H++ server and the results are discussed. In conclusion, one or more of Cys residues interact and involved in the pKa value of the active site Glu-376. It could thus be concluded that Cys residue(s) has crucial role(s) in the conformation stability and biocatalytic activity of the IBD.

show abstract

Section: Fig (1)mentioning

confidence: 63%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Stopped-flow kinetics and cysteine residue protonation of human isobutyryl-CoA dehydrogenase

2017

Egyptian Journal of Pure and Applied Science

View full text Add to dashboard Cite

show abstract

“…IBD was purified using a modification of the published protocols [29,30]. The cells producing IBD, following IPTG induction for 3 hrs, was subjected to sonication in 100 mM Tris buffer containing 150 mM EDTA, pH 8.…”

Section: Methodsmentioning

confidence: 99%

Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands

Mohsen

Vockley

2015

Biochimie

View full text Add to dashboard Cite

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and the transfer of electrons to the electron transfer flavoprotein (ETF). Recombinant human IVD purifies with bound CoA-persulfide. A modified purification protocol was developed to isolate IVD without bound CoA-persulfide and to protect the protein thiols from oxidation. The CoA-persulfide-free IVD specific activity was 112.5 µmol porcine ETF•min−1•mg−1, which was ~20-fold higher than that of its CoA-persulfide bound form. The Km and catalytic efficiency (kcat/Km) for isovaleryl-CoA were 1.0 µM and 4.3 × 106•M−1•sec−1 per monomer, respectively, and its Km for ETF was 2.0 µM. Anaerobic titration of isovaleryl-CoA into an IVD solution resulted in a stable blue complex with increased absorbance at 310 nm, decreased absorbance at 373 and 447 nm, and the appearance of the charge transfer complex band at 584 nm. The apparent dissociation constant (KD app) determined spectrally for isovaleryl-CoA was 0.54 µM. Isovaleryl-CoA, acetoacetyl-CoA, methylenecyclopropylacetyl-CoA, and ETF induced CD spectral changes at the 250–500 nm region while isobutyryl-CoA did not, suggesting conformational changes occur at the flavin ring that are ligand specific. Replacement of the IVD Trp166 with a Phe did not block IVD interaction with ETF, indicating that its indole ring is not essential for electron transfer to ETF. A twelve amino acid synthetic peptide that matches the sequence of the ETF docking peptide competitively inhibited the enzyme reaction when ETF was used as the electron acceptor with a Ki of 1.5 mM.

show abstract

Acyl‐CoA dehydrogenase substrate promiscuity: Challenges and opportunities for development of substrate reduction therapy in disorders of valine and isoleucine metabolism

Dodatko

Dwyer

Violante

et al. 2023

J of Inher Metab Disea

View full text Add to dashboard Cite

Toxicity of accumulating substrates is a significant problem in several disorders of valine and isoleucine degradation notably short‐chain enoyl‐CoA hydratase (ECHS1 or crotonase) deficiency, 3‐hydroxyisobutyryl‐CoA hydrolase (HIBCH) deficiency, propionic acidemia (PA), and methylmalonic aciduria (MMA). Isobutyryl‐CoA dehydrogenase (ACAD8) and short/branched‐chain acyl‐CoA dehydrogenase (SBCAD, ACADSB) function in the valine and isoleucine degradation pathways, respectively. Deficiencies of these acyl‐CoA dehydrogenase (ACAD) enzymes are considered biochemical abnormalities with limited or no clinical consequences. We investigated whether substrate reduction therapy through inhibition of ACAD8 and SBCAD can limit the accumulation of toxic metabolic intermediates in disorders of valine and isoleucine metabolism. Using analysis of acylcarnitine isomers, we show that 2‐methylenecyclopropaneacetic acid (MCPA) inhibited SBCAD, isovaleryl‐CoA dehydrogenase, short‐chain acyl‐CoA dehydrogenase and medium‐chain acyl‐CoA dehydrogenase, but not ACAD8. MCPA treatment of wild‐type and PA HEK‐293 cells caused a pronounced decrease in C3‐carnitine. Furthermore, deletion of ACADSB in HEK‐293 cells led to an equally strong decrease in C3‐carnitine when compared to wild‐type cells. Deletion of ECHS1 in HEK‐293 cells caused a defect in lipoylation of the E2 component of the pyruvate dehydrogenase complex, which was not rescued by ACAD8 deletion. MCPA was able to rescue lipoylation in ECHS1 KO cells, but only in cells with prior ACAD8 deletion. SBCAD was not the sole ACAD responsible for this compensation, which indicates substantial promiscuity of ACADs in HEK‐293 cells for the isobutyryl‐CoA substrate. Substrate promiscuity appeared less prominent for 2‐methylbutyryl‐CoA at least in HEK‐293 cells. We suggest that pharmacological inhibition of SBCAD to treat PA should be investigated further.

show abstract

Biochemical Studies on Recombinant Human Isobutyryl-CoA Dehydrogenase

Cited by 4 publications

References 24 publications

Stopped-flow kinetics and cysteine residue protonation of human isobutyryl-CoA dehydrogenase

Stopped-flow kinetics and cysteine residue protonation of human isobutyryl-CoA dehydrogenase

Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands

Acyl‐CoA dehydrogenase substrate promiscuity: Challenges and opportunities for development of substrate reduction therapy in disorders of valine and isoleucine metabolism

Contact Info

Product

Resources

About