Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii

Abstract

Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii has been purified and crystallized, and some of its properties have been established. The work was facilitated by a newly developed coupled and continuous spectrophotometric assay in which the disappearance of added acrylate could be followed at 245 nm. The rate-limiting conversion of acetyland f,-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to f3-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated f3-hydroxypropionyl CoA. This product then served to generate more acrylyl CoA in cyclic fashion. The net result was the CoA transferase-limited conversion of acrylate to ,B-hydroxypropionate. The purified transferase has a molecular weight of 125,000 and is composed of two subunits of 63,000 each, as determined by disc gel electrophoresis. Short-chain-length monocarboxylic acids are substrates, whereas dicarboxylic or f3-ketocarboxylic acids are not. The reaction kinetics are typical of a ping-pong bi bi mechanism composed of two half reactions linked by a covalent enzyme intermediate. Incubation of the transferase with acetyl CoA in

The Reversible Dehydration of (R)‐2‐Hydroxyglutarate to (E)‐Glutaconate

“…Probably, the acids are activated by a specific CoA-transferase similar to that from M . elsrlenii [5]. If this activation indeed occurs, one should consider an isomerase or an epimerase attacking only the thiol esters and not the free acids.…”

“…However the existence of this reaction was questioned recently [5]. This paper will show that in the case of (R)-2-hydroxyglutarate this type of reaction indeed proceeds as outlined above [Eqn(l)].…”

“…The dehydration was inhibited by 2 mM azide, 1 mM arsenate, 1 mM hydroxylamine, 20 pM dinitrophenol or 10 pM carbonylcyanide p-trifluoromethoxyphenylhydrazone. 5. It is concluded that the actual substrates of the dehydration are the corresponding thiol esters.…”

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids

“…Different types of CoA transferase have been subsequently shown to be present in several other species of bacteria. These enzymes have been prepared in very pure form from Escherichia coli (24), Peptostreptococcus elsdenii (28), and Clostridium sp. strain SB4 (3).…”

Unusual dehydrations in anaerobic bacteria

“…However, /3lactate (3-bydroxypropionate) and 3-hydroxyglutarate were not fermented by cell-free extracts of C. propionicum [14] and ,4. fermentans [44], respectively, whereas (R)-iactate and 2-hydroxyglutarate were fermented readily. The 3-hydroxy acids were certainly activated to the CoA esters under these conditions since the corresponding CoA-transferases do not exhibit high specificities [14,48,60]. Thus 3-hydroxyacyl-CoA esters are no substrates for the corresponding 2-hydroxyacyl-CoA dehydratases.…”