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Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects isoleucine catabolism and ketone body metabolism. We identified 7 novel and 2 previously reported mutations in six T2-deficient patients. Transient expression analysis of wild-type and eight mutant cDNAs was performed at 40, 37 and 30 degrees C. Although no significant residual activity was detected, mutant proteins were detected in the N158D, N158S, R208Q, Y219H and N282H mutants. Accumulation of these mutant proteins was temperature-sensitive with the highest expression levels at lower temperatures. Expression of Q73P and N353K cDNAs yielded neither residual T2 protein nor enzyme activity. An E252del mutant T2 was detected with a relative protein amount and enzyme activity of 30% and 25%, respectively, in comparison to the wild-type at 37 degrees C. The E252del mutant protein was more stable at 30 degrees C expression than 37 degrees C, but was essentially undetectable at 40 degrees C, indicating its temperature-sensitive instability. Kinetic studies revealed a twofold K(m) elevation for substrates coenzyme A and acetoacetyl-CoA in the E252del mutant, while V(max) was comparable to the wild-type. We conclude that the E252del is a temperature-sensitive K(m) mutant. This correlates well with the effect predicted from the T2 tertiary structure analysis, using the crystal structure of the human T2 homotetramer. The probable effect of the other mutations on the T2 tertiary structure was also evaluated.