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

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Abstract
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.