Identification of glutamate beta 54 as the covalent-catalytic residue in the active site of glutaconate CoA-transferase from Acidaminococcus fermentans.

In the course of glutamate fermentation by Acidaminococcus fermentans glutaconate coenzyme A-transferase catalyzes the transfer of CoAS- from acetyl-CoA to (R)-2-hydroxyglutarate, forming (R)-2-hydroxyglutaryl-CoA. Glutamate (E) 54 of the beta-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, beta E54A, beta E64A, beta E54Q and beta E54D, were introduced by mismatch oligonucleotide priming. As expected, beta E54A was inactive (0.02% of the wild-type), whereas beta E64A and beta E54D 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 beta E54Q mutant increased from 1% to almost 100% upon incubation with acetyl-CoA and glutaconate at 37 degrees C within 40 h. Hence, the substrates induced the conversion of the mutant glutamine residue into the glutamate residue of the wild-type enzyme.