Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii.
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 acetyl- and beta-hydroxypropionyl CoA to acrylyl CoA by CoA transferase was followed by the non-rate-limiting conversion to beta-hydroxypropionyl CoA by excess crotonase. Thus, a small priming quantity of acetyl CoA served to generate acrylyl CoA, which, by hydration, generated beta-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 beta-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 beta-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 absence of a fatty acid acceptor yielded a stable intermediate which, by absorption spectrophotometry, radioactivity measurements, reduction with borohydride, reactivity with hydroxylamine, and catalytic activity, was identified as an enzyme-CoA compound. Kinetic constants for CoA transferase are: final specific activity, 110 U/mg of protein corresponding to 1.38 X 10(4) mumol of acrylate activated per mumol of transferase; Km for acrylate, 1.2 X 10(-3) M; Km for acetyl CoA (beta-hydroxypropionyl CoA), 2.4 X 10(-5) M.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000179:Acrylates / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D002264:Carboxylic Acids / Q000494:pharmacology; D002474:Cell-Free System; D003065:Coenzyme A; D003437:Crotonates; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010462:Peptostreptococcus / Q000201:enzymology*; D011422:Propionates / Q000096:biosynthesis; D014166:Transferases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism
D000179:Acrylates; D000596:Amino Acids; D002264:Carboxylic Acids; D003437:Crotonates; D011422:Propionates; D000105:Acetyl Coenzyme A; D014166:Transferases; D006867:Hydrolases; D003065:Coenzyme A