Purification and characterization of 2-methyl-branched chain acyl coenzyme A dehydrogenase, an enzyme involved in the isoleucine and valine metabolism, from rat liver mitochondria.

Y Ikeda,K Tanaka

doi:10.1016/s0021-9258(17)44692-8

Abstract

2-Methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis both with and without 2-mercaptoethanol, the enzyme showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size. Its isoelectric point was 5.50 +/- 0.2, and A1%280 nm was 12.5. This enzyme contained protein-bound FAD. The purified enzyme dehydrogenated S-2-methylbutyryl-CoA and isobutyryl-CoA with equal activity. The activities with each of these compounds were co-purified throughout the entire purification procedure. This enzyme also dehydrogenated R-2-methylbutyryl-CoA, but the specific activity was considerably lower (22%) than that for the S-enantiomer. The enzyme did not dehydrogenate other acyl-CoAs, including isovaleryl-CoA, propionyl-CoA, butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA, at any significant rate. Apparent Km and Vmax values for S-2-methylbutyryl-CoA were 20 microM and 2.2 mumol min-1 mg-1, respectively, while those for isobutyryl-CoA were 89 microM and 2.0 mumol min-1 mg-1 using phenazine methosulfate as an artificial electron acceptor. The enzyme was also active with electron transfer flavoprotein. Tiglyl-CoA and methacrylyl-CoA were identified as the reaction products from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced from R-2-methylbutyryl-CoA. Tiglyl-CoA competitively inhibited the activity with both S-2-methylbutyryl-CoA and isobutyryl-CoA with a similar Ki. The enzyme activity was also severely inhibited by several organic sulfhydryl reagents such as N-ethylmaleimide, p-hydroxymercuribenzoate, and methyl mercury iodide. The pattern and degree of inhibition were essentially identical for both substrates. The purified 2-methyl-branched chain acyl-CoA dehydrogenase was immunologically distinct from isovaleryl-CoA-, short chain acyl-CoA-, medium chain acyl-CoA-, or long chain acyl-CoA dehydrogenase.

Highlights

In thecourse of these studies,we demonstrated for the first acrylyl-CoA were identified as the reaction products time that 2-methylbutyryl-CoA and isobutyryl-CoA were not from S-2-methylbutyryl-CoA and isobutyryl-CoA, respectively. 2-Ethylacrylyl-CoA was produced fromR2-methylbutyryl-CoA
Technical details aredescribed under "Experimen- further eluted with 10 mM KPO, buffer containing tal Procedures." The first four steps of the purification are 0.8 M NaCl and 3 mM FAD, the butyryl-CoA- and long chain the same as previously described [8].Rat liver mitochondria acyl-CoA dehydrogenases which still remained in the column were solubilized by sonication and the supernatant were elutedas a sharppeak (Fig. 1)
Were 7 PM whenS-2-methylbutyryl-CoA was the substrate and 3 p~ whenisobutyryl-CoA was thesubstrate.These results indicate that tiglyl-CoA, the product from S-2-methylbutyryl-CoA, competitively inhibits the dehydrogenationof both S-2-methylbutyryl-CoA anisdobutyryl-CoA with similar

Summary

Introduction

Technical details aredescribed under "Experimen- further eluted with 10 mM KPO, buffer (pH 8.0) containing tal Procedures." The first four steps of the purification are 0.8 M NaCl and 3 mM FAD, the butyryl-CoA- and long chain the same as previously described [8].Rat liver mitochondria acyl-CoA dehydrogenases which still remained in the column were solubilized by sonication (step 1) and the supernatant were elutedas a sharppeak (Fig. 1).

Results

Conclusion