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Abstract
Mitochondrial acetoacetyl‐CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine‐derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2‐deficient patients. The 56 disease‐associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease‐associated missense ACAT1 variants have been measured by expressing them in human SV40‐transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild‐type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening.
Highlights
The mitochondrial acetoacetyl‐CoA thiolase is a ubiquitous and important enzyme for ketone body synthesis and degradation as well as in isoleucine catabolism (Fukao et al, 2014, 2018)
Functional studies of 30 disease‐associated missense mitochondrial acetoacetyl‐CoA thiolase aDescription of nucleotide changes (T2) variants have been performed in vitro, using the potassium ion‐activated acetoacetyl‐CoA degradation assay and for all these variants low activity is observed (Table 1)
Patients with mild variants can develop episodic ketoacidosis as severe and frequent as those with severe variants (Fukao et al, 2001). This raises questions whether the T2 activity measured in vitro using acetoacetyl‐CoA as a substrate fully reflects the in vivo T2 deficiency, and whether it is better to use 2‐methylacetoacetyl‐CoA as specific substrates
Summary
The mitochondrial acetoacetyl‐CoA thiolase (commonly known as β‐ketothiolase [T2]; EC 2.3.1.9; encoded by the ACAT1 gene) is a ubiquitous and important enzyme for ketone body synthesis and degradation as well as in isoleucine catabolism (Fukao et al, 2014, 2018). At least, five other thiolase isoenzymes: Cytosolic acetoacetyl‐CoA thiolase (CT, EC 2.3.1.9), mitochondrial 3‐ketoacyl‐CoA thiolase (T1, EC 2.3.1.16), the β subunit of the mitochondrial trifunctional enzyme that catalyzes 3‐ketoacyl‐CoA thiolase activity (TFE, EC 2.3.1.16), the peroxisomal 3‐ketoacyl‐CoA thiolase (AB‐thiolase, EC 2.3.1.16), and the peroxisomal thiolase type‐1 (SCP2‐thiolase; EC 2.3.1.176) These thiolases (excluding the SCP2‐ thiolase) share 35–46% sequence identity and have both synthetic and degradative functions; the degradative SCP2‐thiolase has very low sequence similarity with any of the other thiolase family members. The functional site of each of the subunits of the T2 tetramer is very extensive, including the catalytic residues and the residues that shape the CoA binding site Enzyme assayd/Expression assaye by expression at Important properties of each residue with respect to the structure of the tetramerf: Nucleotide E/I changea
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