Stereochemistry of propionyl-coenzyme A and pyruvate carboxylations catalyzed by transcarboxylase.
The stereochemistry of the two half-reactions catalyzed by the biotin-containing enzyme, transcarboxy-lase from Propionobacteria shermanii, has been determined. The pro-R hydrogen at C-2 of propionyl-coenzyme A is replaced by CO2 in formation of the S isomer of methylmalonyl-CoA, defining the process as retention of configuration. This C-2 hydrogen is abstracted at a rate identical with product formation. For the other half-reaction, pyruvate to oxalacetate, the chiral methyl group methodology of Rose (I. A. Rose (1970), J. Biol. Chem. 245, 6052) was employed. First, it was determined with [3-2-He]pyruvate that a kinetic deuterium isotope effect of 2.1 occurs at Vmax in this carboxyl transfer, indicating that the necessary requirement for discrimination against heavy isotopes of hydrogen existed. Then, 3(S)-[3-2-H,3-H]pyruvate, generated from 3(S)-]E-2-H,3-H]phosphoglycerate, was carboxylated and the oxalacetate trapped as [3030H]malate using malate dehydrogenase. Exhaustive incubation of the tritiated malate (3-H/14-C = 1.95) with fumarase to labilize the pro-R hydrogen at C-3 resulted in release of 65% of the tritium into water. Reisolation of the malate after fumarase action yielded a 30H/14-C ration of 0.67, indicating 34% retention as expected. The theoretical enantiotopic distribution for the observed k1H/k2H of 2.1 is 68:32. Selective enrichment of tritium in the pro-R position at C-3 of malate indicates enzymatic carboxylation of pyruvate with retention of configuration in this half-reaction also.
D002262:Carboxy-Lyases / Q000032:analysis; D019878:Carboxyl and Carbamoyl Transferases; D003065:Coenzyme A / Q000031:analogs & derivatives*; D003903:Deuterium; D007700:Kinetics; D008293:Malates; D008764:Methylmalonic Acid; D008956:Models, Chemical; D011422:Propionates; D011424:Propionibacterium / Q000201:enzymology; D011773:Pyruvates / Q000378:metabolism; D013237:Stereoisomerism; D014166:Transferases / Q000032:analysis
D008293:Malates; D011422:Propionates; D011773:Pyruvates; D008764:Methylmalonic Acid; D003903:Deuterium; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D002262:Carboxy-Lyases; D003065:Coenzyme A