Substrate specificities of 3-oxoacyl-CoA thiolase A and sterol carrier protein 2/3-oxoacyl-CoA thiolase purified from normal rat liver peroxisomes. Sterol carrier protein 2/3-oxoacyl-CoA thiolase is involved in the metabolism of 2-methyl-branched fatty acids and bile acid intermediates.
The two main thiolase activities present in isolated peroxisomes from normal rat liver were purified to near homogeneity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the first enzyme preparation displayed a single band of 41 kDa that was identified as 3-oxoacyl-CoA thiolase A (thiolase A) by N-terminal amino acid sequencing. The second enzyme preparation consisted of a 58- and a 46-kDa band. The 58-kDa polypeptide reacted with antibodies raised against either sterol carrier protein 2 or the thiolase domain of sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCP-2/thiolase), formerly also called sterol carrier protein X, whereas the 46-kDa polypeptide reacted only with the antibodies raised against the thiolase domain. Internal peptide sequencing confirmed that the 58-kDa polypeptide is SCP-2/thiolase and that the 46-kDa polypeptide is the thiolase domain of SCP-2/thiolase. Thiolase A catalyzed the cleavage of short, medium, and long straight chain 3-oxoacyl-CoAs, medium chain 3-oxoacyl-CoAs being the best substrates. The enzyme was inactive with the 2-methyl-branched 3-oxo-2-methylpalmitoyl-CoA and with the bile acid intermediate 24-oxo-trihydroxycoprostanoyl-CoA. SCP-2/thiolase was active with medium and long straight chain 3-oxoacyl-CoAs but also with the 2-methyl-branched 3-oxoacyl-CoA and the bile acid intermediate. In peroxisomal extracts, more than 90% of the thiolase activity toward straight chain 3-oxoacyl-CoAs was associated with thiolase A. Kinetic parameters (Km and Vmax) were determined for each enzyme with the different substrates. Our results indicate the following: 1) the two (main) thiolases present in peroxisomes from normal rat liver are thiolase A and SCP-2/thiolase; 2) thiolase A is responsible for the thiolytic cleavage of straight chain 3-oxoacyl-CoAs; and 3) SCP-2/thiolase is responsible for the thiolytic cleavage of the 3-oxoacyl-CoA derivatives of 2-methyl-branched fatty acids and the side chain of cholesterol.
Antonenkov
V D
VD
Katholieke Universiteit Leuven, Departement Moleculaire Celbiologie, Afdeling, Campus Gasthuisberg (O & N), Herestraat 49, B-3000 Leuven, Belgium.
Van Veldhoven
P P
PP
Waelkens
E
E
Mannaerts
G P
GP
D000101:Acetyl-CoA C-Acetyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005227:Fatty Acids / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D008297:Male; D008745:Methylation; D008830:Microbodies / Q000737:chemistry / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D013054:Spectrophotometry, Atomic; D013261:Sterols / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D005227:Fatty Acids; D013261:Sterols; C072087:sterol carrier proteins; D000101:Acetyl-CoA C-Acetyltransferase