CoA (11)
and was not tested with acetoacetyl-CoA in this experiment]. On the other hand, when the thiolase activities were measured in parallel by the formation of new ester bonds and by the disappearance of the Mg 2Ο© -enolate complex, rates of the latter reaction were still seriously lower than the rates of new ester bond formation, when the short-to medium-chain substrates were used (C 6 and C 7 for thiolase A; C 6 , C 7 , and C 8 for SCP-2/thiolase; data not shown). Similarly, the stoichiometry of acetoacetyl-CoA cleavage (48 mol acetyl-CoA formed/min β
mg protein) by thiolase A in the presence of 5 mM Mg 2Ο© closely agreed with the formation of new ester bonds (26 mol/min β
mg protein), but the rate of disappearance of the Mg 2Ο© -enolate complex was drastically lower (11 mol/min β
mg protein). When 3-oxooctanoyl-CoA was used, the results of all three assays for thiolase A approximately coincided (98, 87, and 115 mol/min β
mg protein for thioester bond formation, acetyl-CoA production, and Mg 2Ο© -enolate complex disappearance, respectively). These data are difficult to explain in terms only of an inhibitory effect of Mg 2Ο© on the thiolytic cleavage reaction. They imply that the cleavage of 3-oxoacyl-CoAs (C 4 -C 7 ) by thiolase A as well as that of medium-chain 3-oxoacyl-CoAs by SCP-2/thiolase may not be the rate-limiting step in the case of the Mg 2Ο© -enolate assay.
Our data clarify the contradictory results related to the effect of Mg 2Ο© on thiolase activity obtained by different authors (3, 5-10) and also indicate that the commonly used Mg 2Ο© -enolate assay leads to an underestimation of the thiolase activity, especially toward short-and medium-chain 3-oxoacyl-CoAs.