Subunit composition of oxaloacetate decarboxylase and characterization of the α chain as carboxyltransferase

Abstract

Oxaloacetate decarboxylase from Klebsiella aerogenes was shown to be composed of three different subunits a, b, y with M , 65 000, 34000 and 12000, respectively. On dodecylsulfate/polyacrylamide gels the smallest of these subunits was heavily stained with silver but poorly with Coomassie brilliant blue. All three subunits were resolved and clearly detectable by high-performance liquid chromatography in a dodecylsulfate-containing buffer. Biotin was localized exclusively in the a chain.Freezing and thawing of the isolated membranes in the presence of 1 M LiCl released the a chain which was subsequently purified to near homogeniety by affinity chromatography on monomeric avidin-Sepharose. No p or y chain were detectable in this a chain preparation and no oxaloacetate decarboxylation was catalyzed. The isolated a chain, however, was a catalytically active carboxyltransferase as evidenced from the isotopic exchange between [l-'4C]pyruvate and oxaloacetate. The rate of this exchange reaction was about 9 U/mg protein and was completely independent of the presence of N a + ions. The ease with which the a chain was released from the membrane characterize this subunit as a peripheral membrane protein. The p and y chain, on the other hand, stick so firmlyin the membrane that they are only released by detergents, thus indicating that these are integral membrane proteins. Limited tryptic digestion of oxaloacetate decarboxylase led to a rapid cleavage of the a chain, yielding a polypeptide of M , 52 000 which was devoid of biotin. Degradation of the p chain required prolonged incubation periods and was markedly influenced by Na' ions which had a protective effect against proteolysis. A proton is required in the decarboxylation of oxaloacetate and C 0 2 arises as primary product. The other alternative, i. e. generation of HCO; with H 2 0 as substrate, has been excluded.The sodium transport decarboxylases constitute a unique group of vectorial catalysts converting the chemical energy of decarboxylation reactions into electrochemical gradients of Na [6] were also shown to be sodiumtransport decarboxylases. These three enzymes are remarkably similar in many properties, e. g. in their localization in the cell membrane, the specific activation by Na' ions and in containing biotin as a prosthetic group. Carboxylation of this prosthetic group by carboxyl transfer is the first step in the overall reaction and has been demonstrated for oxaloacetate decarboxylase to be independent of Na' ions [7]. The N1-carboxybiotin enzyme intermediate is subsequently decarboxylated in an Naf-dependent reaction and this drives the uphill transport of Na' ions through the membrane.The biotin prosthetic group of the sodium-transport decarboxylases has tremendously facilitated the purification of these enzymes which is achieved by affinity chromatography on monomeric avidin-Sepharose columns [5, 81. Dodecylsulfate

Kinetic analysis of the reaction mechanism of oxaloacetate decarboxylase from Klebsiella aerogenes

“…Carboxylases perform the carboxylation of biotin under ATP hydrolysis and decarboxylases catalyze the decarboxylation of carboxybiotin, thereby generating an Na + concentration gradient. Under certain conditions, the direction of these reactions is reversed, i. e. ATP is formed under decarboxylation of carboxybiotin [I21 and biotin is carboxylated with the energy of an Na' gradient [5]. Thus, carboxylases can in principle act as decarboxylases and decarboxylases can act as carboxylases.…”

“…The a chain is a catalytically active carboxyltransferase but has no decarboxylase activity which is therefore probably catalyzed by another subunit [5]. This could be the / I chain to which Na+ ions, the substrate for the decarboxylation, are specifically bound [5].…”

“…This step requires Na' ions for catalytic activity and is coupled to the vectorial translocation of Na' ions through the cell membrane by membrane-bound oxaloacetate decarboxylase (Eqn 3) [3] whereas obviously no physical change of Na' occurs with the soluble enzyme. The fl and/or y subunit are required for the decarboxylation of the carboxybiotinenzyme and therefore probably contain the catalytic site for this partial reaction [5].…”

“…The enzyme is composed of three different polypeptide chains 01, j?, y with M, of 65000,34000 and 12000, respectively [5]. The a chain, containing the biotin prosthetic group, is a peripheral membrane protein whereas p and y subunits are integral membrane proteins.…”

“…The first step is a carboxyl transfer from oxaloacetate to the biotin prosthetic group (Eqn 1). The freely reversible reaction is catalyzed in the absence of Na' ions from the enzyme complex [6] or from the isolated a chain which therefore is the carboxyltransferase subunit [5]. The carboxybiotin-enzyme is decarboxylated in the second step (Eqn 2) to yield C 0 2 and free biotin-enzyme [6].…”