Bovine kidney 3-methylcrotonyl-CoA and propionyl-CoA carboxylases: Each enzyme contains nonidentical subunits

Abstract

3-Methylcrotonyl-CoA carboxylase (MCase; EC 6.4.1.4) and propionyl-CoA carboxylase (PCase; EC 6.4.1.3) have been obtained in highly purified form from bovine kidney mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that each enzyme is composed of nonidentical subunits, including a smaller biotin-free subunit (M, 62,000 and 58,000 for MCase and PCase, respectively), and a larger biotin-containing subunit (M, 80,000 and 74,000 for MCase and PCase, respectively). The possibility that these subunits were derived from a single, larger precursor polypeptide via proteolysis was explored by purification and electrophoresis of each enzyme in the presence of protease inhibitors, but no evidence for proteolysis was obtained. Specific antisera directed towards each enzyme were prepared. The anti-PCase preparation was used to precipitate crossreacting PCase from a pig heart extract. Analysis of the immunoprecipitate obtained revealed a biotin-containing polypeptide (M, 78,000) and a biotin-free polypeptide (Mr 55,000), suggesting that pig heart PCase also contains nonidentical subunits analogous to those seen in the kidney mitochondrial MCase and PCase. A bipartite subunit structure may be a common feature in mammalian MCase and PCase.Deficiencies in two biotin-containing enzymes, propionyl-CoA carboxylase (PCase; EC 6.4.1.3) and 3-methylcrotonyl-CoA carboxylase (MCase; EC 6.4.1.4), are responsible for the genetically inherited diseases, propionicacidemia and 3-methylcrotonylglycinuria, respectively, in humans (see refs. 1-4 for a review). In order to determine the structural defect(s) in the dysfunctioning enzymes, basic information on the structure of the corresponding normal enzymes is needed. We have been working to obtain purified PCase and MCase from a convenient animal source for such studies. Bovine kidney mitochondria represent a rich source of MCase (unpublished data) and of PCase, and we have recently obtained highly purified preparations of both enzymes. Based on published properties of crystalline pig heart PCase (5) and in analogy with other biotin enzymes from animal tissues, including acetyl-CoA carboxylase (6)(7)(8) and pyruvate carboxylase (9), we had assumed that PCase and MCase from animal sources would each contain one type of multifunctional subunit with all three catalytic subsites fused into a single polypeptide chain (see refs. 10 and 11 for a review). We report here the unexpected finding that mitochondrial MCase and PCase both contain two different types of subunits, a structural pattern of biotin enzymes heretofore ascribed only to bacterial biotin enzymes (10-12).t The significance of this finding in relation to the evolution of biotin enzymes and to the interpretation of recent genetic studies on human PCase deficiency will be discussed.EXPERIMENTAL PROCEDURE Enzyme Purification. MCase and PCase were purified from bovine kidney mitochondria as outlined below. Mitochondria, isolated by differential centrifugation (15), were disrupted w...

Propionyl-CoA carboxylase – A review

“…Biotin is an obligate co-factor [2–5]. PCC is part of a subgroup of small acyl CoA carboxylases (YCC) which also includes acetyl CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, and geranyl- CoA carboxylase [6].…”

“…The PCCA precursor does not contain biotin until imported into the mitochondrion and cleaved [2, 3, 9]. Biotin is loaded onto lysine 669 by holocarboxylase synthase (*60918; E.C.…”

“…Propionyl-CoA is produced by catabolism of c holesterol, v aline, o dd chain fatty acids, m ethionine, i soleucine and t hreonine (c-VOMIT) [2, 16–18]. Like other carboxylases, PCC is promiscuous and can carboxylate several other acyl-CoAs, but has greatest affinity for propionyl-CoA (Km = 0.29 mM).…”

The Use of Avidin as a Probe for the Distribution of Mitochondrial Carboxylases in Developing Chick Retina

“…Pyruvate carboxylase, methylcrotynyl CoA carboxylase, and propionyl CoA carboxylase are multi-subunit enzymes involved in the production of oxaloacetate, the degradation of leucine, and the breakdown of methionine, leucine, and fatty acids, respectively (Wood and Barden 1977;Lau et al 1979;Bramwell 1987). The biotinylated subunits (approximate molecular masses of 130, 75, and 72 kD, respectively) of all three enzymes have been localized to the mitochondria of several mammalian tissues and cell lines, by several methods of centrifugation (Moss and Lane 1971;Mackall and Lane 1977;Petrelli et al 1978;Chandler and Ballard 1986) and by immunohistochemical analysis (Gratzner et al 1980).…”

Biotin-response Organicaciduria

“…In fact, this would have been expected to be the predominant class, since all of the other complementation groups would have resulted from the "rare" case of intragenic complementation. Thus, with the mapping of some 23 pcc inutants, the absence of the pccABC class strongly suggests that pccA and pccBC represent two separate genes, perhaps encoding different subunits of PCC (21)(22)(23).…”