Thiolase subunits

The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain. For fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associated with the inner mitochondrial membrane, the trifunctional protein (TFP). TFP is a heterooctamer of 4/34 subunits. Each a subunit contains two activities, the enoyl-CoA hydratase and the /3-hydroxyacyl-CoA dehydrogenase the /3 subunits contain the thiolase activity. This tight association of three enzymes may allow efficient substrate channeling from one active site to the... 

Early in the study of the enzymology of the biosynthesis of HMG-CoA, there was some confusion as to whether these processes were physically separated within the cell. Studies by Lane and his collaborators [13] and by others [14] clearly indicated a duality of locus. The cytosolic enzyme, purified from avian liver [13], was found to have a molecular weight of 1.7 x 10 with 4 apparently identical subunits (41000 by SDS gel electrophoresis). The cytosolic enzyme constituted 70% of total thiolase found in chicken liver. 

Brief incubation of the enzyme at 0°C with radioactive acetyl-CoA led to the formation of acyl enzyme which could be isolated by chromatography on Sephadex [24]. The enzyme-substrate complex was then reacted with acetoacetyl-CoA with the concomitant formation of HMG-CoA. Further studies indicated that the functional group on the enzyme that accepted the acetyl residue was a cysteine sulfhydryl. 4 -Phosphopantetheine is known to accept acyl residues, but it was not found in this protein. The stoichiometry for acetylation was 0.7 acetyl groups per mole of enzyme since it is a dimeric protein with apparently identical subunits, this observation is surprising. Thus, it is possible that the subunits perform different functions for example, one could be regulatory. It is interesting to note that both the thiolase and HMG-CoA synthetase utilize acyl enzyme intermediates in their catalytic mechanisms. 

The negative charge of glutamate-462 was found to be necessary for increasing the thermostability of the multienzyme complex, and amidation of the T carboxyl group of glutamate-462 is known to have an adverse effect on the 3-ketoacyl-CoA thiolase activity associated with the small subunit of the fatty acid oxidation complex.These findings provided evidence that a Glu Gin mutation of mitochondrial trifunctional P-oxidation complex, which corresponds to the Glu -> Gin mutation described above,... 

The 3-oxoacyl-CoA thiolase of cucumber glyoxysomes consists of two identical subunits with a molecular weight of 45000. The protein has an isoelectric point of pH 8.5. Data on the substrate specificity of the enzyme have not yet been reported. 

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