Dimeric pig heart succinate-coenzyme A transferase uses only one subunit to support catalysis

Pig heart succinate-coenzyme A transferase (succinyl-coenzyme A: 3-oxoacid coenzyme A transferase; E. C. 2.8.3.5.), a dimeric enzyme purified by affinity chromatography on Procion Blue MX-2G Sepharose, reacts with acetoacetyl-coenzyme A to form a covalent enzyme-coenzyme A thiolester intermediate in which the active site glutamate (E344) of both subunits each forms thiolester links with coenzyme A. Reaction of this dimeric enzyme-coenzyme A species with sodium borohydride leads to inactivation of the enzyme and reduction of the thiolester on both subunits to the corresponding enzyme alcohol, as judged by electrospray mass spectrometry. Reaction of the dimeric enzyme-coenzyme A intermediate with either succinate or acetoacetate, however, results in only one-half of the coenzyme A being transferred to the acceptor carboxylate to form either succinyl-coenzyme A or acetoacetyl-coenzyme A. Reaction of this latter enzyme species with borohydride caused no loss of enzyme activity despite the reduction of the remaining half of the enzyme-coenzyme A thiolester to the enzyme alcohol. That this catalytic asymmetry existed between subunits within the same enzyme dimer was demonstrated by showing that the enzyme species, created by successive reaction with acetoacetyl-coenzyme A and succinate, bound to Blue MX-2G Sepharose through the remaining available active site and could be eluted as a single chromatographic species by succinyl-coenzyme A. It is concluded that while both of the subunits of the succinate-coenzyme A transferase dimer are able to form enzyme-coenzyme A thiolester intermediates, only one subunit is competent to transfer the coenzyme A moiety to a carboxylic acid acceptor to form the new acyl-coenzyme A product. The possible structural basis for this catalytic asymmetry and its mechanistic implications are discussed

Stereochemical course of the formation of the C(7)-formyl group from a chiral methyl group during the transformation of chlorophyllide a into chlorophyllide b

The biosynthesis of chlorophyll a and chlorophyll b from (2R,3R)- and (2S,3S)-5-amino[2,3-14C2,2,3- 2H2,2,3-3H2]levulinic acid in greening barley has established that chlorophyllide a oxidase catalyses the transformation of the methyl group at C(7) of chlorophyllide a into the CHO group of chlorophyllide b with the loss of HSi from the 7-(hydroxymethyl)chlorophyllide intermediate

Continuous coupled assay for 5-aminolevulinate synthase

This chapter discusses the continuous coupled assay for 5-aminolevulinate synthase. A direct 5-aminolevulinate synthase enzyme assay has been developed on the basis of linking 5-aminolevulinate synthase to pyruvate dehydrogenase. Initial investigations demonstrated that pyruvate dehydrogenase was unaffected by components of the 5-aminolevulinate synthase assay system and vice versa. When 5-aminolevulinate synthase activity was determined using this coupled assay, a linear increase in absorbance was obtained. The rate was dependent on the presence of both succinyl-CoA and glycine and was linear with respect to both time and protein concentration. A Lineweaver–Burk plot of dependence of activity of the Rhodobacter spheroides enzyme on succinyl-CoA gave values for Km and Vmax of 6μM and 138μmol/ mg protein/hour, respectively. The Km for glycine was 5 mM. These values agree well with those in the literature. Data obtained using the coupled assay showed excellent agreement with the data obtained using the discontinuous assay