L-3-Hydroxyacyl Coenzyme A Dehydrogenase from Pig Heart Muscle: II. SUBUNIT STRUCTURE

Abstract

Abstract The molecular weight of 3-hydroxyacyl-CoA dehydrogenase, as measured by sedimentation equilibrium analysis, was found to be 65,000, in good agreement with the value of 67,000 obtained from amino acid analyses. Gel filtration and sedimentation velocity measurements gave a somewhat higher value of 75,000. Further sedimentation equilibrium studies in 6 m guanidine hydrochloride and gel electrophoresis in sodium dodecyl sulfate gave molecular weights of 31,000, indicating that the protein is composed of 2 subunits of equal molecular weight and supporting the lower molecular weight values for the native protein. NH2-terminal analyses gave 2 residues of serine per mole of protein, which is consistent with two polypeptide chains per molecule. Fractionation of the soluble tryptic peptides of the S-[14C]carboxymethylated enzyme by ion exchange chromatography and unidirectional paper electrophoresis indicated a maximum of 46 major peptides, which is approximately one-half of the number of tryptic peptides expected from the lysine plus arginine content of 81. In addition, only one peptide with [14C]carboxymethylcysteine and one peptide containing tryptophan were identified following the fractionation of the soluble tryptic peptides. These observations suggest that the subunits possess very similar or identical amino acid sequences. The sequence of amino acid residues surrounding the single cysteinyl residue is His-Pro-Val-Ser-Cys-Lys. This peptide does not show any obvious relationship to any cysteinyl-containing peptide reported from any other dehydrogenase. The stability of the protein as a function of urea concentration was followed by enzymatic activity, optical rotation, and sedimentation velocity analyses. The molecule appears to undergo a simultaneous dissociation and unfolding with a concomitant complete loss of enzymatic activity between urea concentrations of 2 and 3 m. Approximately 90% of the original activity of enzyme denatured in urea can be recovered upon removal of urea by dialysis against 0.1 m sodium pyrophosphate buffer, pH 7.3, at 4°. Substrate and cofactor do not appear to affect the extent of reactivation under optimal conditions.