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Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase

, : Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase. Protein Science 3(6): 975-981

The enzyme CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase (3-oxoacid CoA transferase), EC is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that its catalytic mechanism involves the transient thioesterification of an active-site glutamate residue by CoA. As a means of identifying this glutamate within the sequence, we have made use of a fortuitous autolytic fragmentation that occurs at the active site when the enzyme-CoA covalent intermediate is heated. The presence of protease inhibitors has no effect on the extent of cleavage detectable by SDS-PAGE, supporting the view that this fragmentation is indeed autolytic. This fragmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the resulting C-terminal fragment is blocked at its N-terminus by a pyroglutamate moiety, it is not amenable to direct sequencing by the Edman degradation method. As an alternative, we have studied a peptide (peptide D) generated specifically by autolysis of the nicked enzyme and predicted to have an N-terminus corresponding to the site of proteolysis and a C-terminus determined by the site of autolysis. This peptide was purified by reversed-phase HPLC and subsequently characterized by electrospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conserved in all sequenced CoA transferases, is the catalytically active amino acid. This information should prove useful to future mutagenesis work aimed at better understanding the active-site structure and catalytic mechanism of CoA transferase.


PMID: 7915164

DOI: 10.1002/pro.5560030613

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Ambrosino L.; Iacono F.; Greco A.; Greco G.M., 1990: Peripheral retinal cryopexy for subtotal vitreous hemorrhage. We used a peripheral retinal cryopexy to treat 15 eyes in 13 patients with subtotal vitreous hemorrhage secondary to proliferative diabetic retinopathy. In some of them complete panretinal photocoagulation had been performed before the hemorrhage....

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Smith Susan L., 1980: Mount Isa; more than a mining town. Lapidary Journal: , Pages 1834-1837. 1980.

Schiner., 1865: Schiner remarks upon Philippi's descriptions of new Chilian Diptera, Verh. zool.-bot. Ges. in Wien,. Schiner remarks upon Philippi's descriptions of new Chilian Diptera, Verh. zool.-bot. Ges. in Wien, 1865, pp. 63-66 (Sitzungsb.).

Chevallier, B.; Fumoleau, P.; Kerbrat, P.; Monnier, A.; Roche, H.; Goudie, M.J.; Herait, P., 1990: Association of bolus tetrahydropyranyl adriamycin and 120 hours continuous 5-fluorouracil infusion in patients with metastatic breast cancer. 4'-O-Tetrahydropyranyl adriamycin (THP adriamycin) is a new anthracycline active as a single drug in advanced breast cancer. We have undertaken a phase II study as first-line treatment for metastatic disease with THP adriamycin day 1 = 40 mg/...

Jordahn, A.C., 1946: Promising new palm introductions. Short descriptions are given of 18 "newer successes" in the palm collection at the Fairchild Tropical Garden, Florida, which contains over 350 species and varieties.

Orozco, G.; Sánchez, E.; González-Gay, M.A.; López-Nevot, M.A.; Torres, Bén.; Pascual-Salcedo, D.; Balsa, A.; Pablos, J.L.; García, A.; González-Escribano, M.Francisca.; Martín, J., 2006: SLC22A4, RUNX1, and SUMO4 polymorphisms are not associated with rheumatoid arthritis: a case-control study in a Spanish population. To replicate the association reported in Japanese individuals of functional SLC22A4 and RUNX1 polymorphisms with rheumatoid arthritis (RA), and to test the possible role in this trait of a functional variant of the SUMO4 gene that was shown to be...

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