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Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

ABSTRACT: Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients.

SUBMITTER: Fukao T

PROVIDER: S-EPMC329848 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Fukao T T Yamaguchi S S Kano M M Orii T T Fujiki Y Y Osumi T T Hashimoto T T

The Journal of clinical investigation 19901201 6

Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothi ...[more]

PMID: 1979337

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Project description:1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg(2+) inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg(2+) but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a K(i) of 67mum. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.

Dataset Information

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Publications

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Similar Datasets

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