Identification of glutamate β54 as the covalent‐catalytic residue in the active site of glutaconate CoA‐transferase from Acidaminococcus fermentans

FEBS Letters

Buckel, Wolfgang¹  Mack, Matthias¹ 

[1] Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität Marburg, Karl-von-Frisch-Str., D-35032 Marburg, Germany

关键词: Glutaconate CoA-transferase;    Site-directed mutagenesis;    βE54 as covalent catalytic residue;    Post-translational activation by the substrates;    Acidaminococcus fermentans;   

DOI  :  10.1016/0014-5793(94)01351-Z

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

In the course of glutamate fermentation by Acidaminococcus fermentans glutaconate coenzyme A-transferase catalyzes the transfer of CoAS⁻ from acetyl-CoA to (R)-2-hydroxyg;utarate, forming (R)-2-hydroxyglutaryl-CoA. Glutamate (E) 54 of the β-subunit was postulated to be directly involved in catalysis by formation of a CoASH ester intermediate [(1994) Eur. J. Biochem., in press]. In order to prove this preliminary result, the following mutations, βE54A, βE64A, βE54Q and βE54D, were introduced by mismatch oligonucleotide priming. As expected, βE54A was inactive (0.02% of the wild-type), whereas βE64A and βE54D were active, 30% and >7%, respectively. However, no CoASH intermediate was detected in the latter mutant, indicating a change in the catalytic mechanism. The activity of the βE54Q mutant increased from 1% to almost 100% upon incubation with acetyl-CoA and glutaconate at 37°C within 40 h. Hence, the substrates induced the conversion of the mutant glutamine residue into the glutamate residue of the wild-type enzyme.

【 授权许可】

Unknown