RESPECTO AL SEGUNDO OBJETIVO ESPECÍFICO

ACC contains a biotin prosthetic group, covalently attached via its carboxyl group to the E-amino group o f a lysine residue on the enzyme. It is one o f four known mammalian biotin-containing enzymes where the biotin group serves as a carrier of activated CO2. The other three biotin-containing enzymes are situated in the

mitochondria (pyruvate carboxylase, propionyl-CoA carboxylase, 3-methyl crotonyl- CoA carboxylase). ACC is the only cytosolic enzyme in this group. The reaction catalysed by ACC can be partitioned into discrete half-reactions (Moss and Lane,

1971).

E-biotin + HCO” + ATP —^E-biotiB-CCf + AD? + Pi E-biotin-Crf -f- acetyl-CoA —► malonyl-CoA+ E-biotin

The initial step involves the MgATP dependent carboxylation o f the biotinyl prosthetic group of the enzyme to form a I'N-carboxybiotinyl enzyme intermediate. In the second step, carboxyl transfer from carboxybiotinyl enzyme to acetyl-CoA gives rise to malonyl-CoA. Both half-reactions are activated by citrate, and enhanced reactivity of the carboxybiotinyl moiety has been correlated with citrate-induced conformational changes at the prosthetic site (Moss and Lane, 1971). In Kcoli, ACC can be resolved into three protein components (Guchait et d , 1974) : a biotin carboxylase, a carboxy transferase and a carboxyl carrier protein which is a non- enzymic protein which contains the covalently bound biotin. Mammalian ACC is a single polypeptide and the model of its domain structure is based on homologies of

the primary amino sequence with carbamoyl phosphate synthetase (catalyses ATP- dependent carboxylation) and propionyl-CoA carboxylase, and the known location of the covalently bound biotin (Haase et d , 1982) (Figure 1.4.). The recent cloning and sequencing o f cDNAs coding for rat and chicken ACC indicates that ACC contains two active sites which have been combined by gene-fusion events (Takai et d , 1988). ACC can be readily cleaved by proteinases to yield two fragments of about 120 KDa (Tanabe et d , 1975; Wada and Tanabe, 1985). Although the site of cleavage has not been defined it is thought to be between the carboxyl carrier and carboxyl transferase domains, where there is an exposed 'hinge region'.

ACC exists as a protomer previously thought of as a homodimer, consisting of two non-covalently associated identical polypeptide chains (Mackall et d , 1978). Mammalian ACC purified from rat liver, adipose tissue and mammary gland is a dimer with a subunit molecular weight of 240 KDa as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 265 KDa as calculated from the polypeptide chain predicted from the cDNA clone (Lopez-Casillas et d ,

1988; Bianchi et d , 1990). ACC is widely distributed in mammalian tissues, especially those active in lipogenesis such as liver, white adipose tissue and mammary gland. However, it also exists in tissues where fatty acid synthesis is not prominent, such as heart and skeletal muscle, where its role is thought to be in regulating fatty acid oxidation (Thampy, 1989). Two isoforms of this enzyme have been identified, a 265 and a 280 KDa isoform (Bianchi et d , 1990; Thampy, 1989; Winz et d , 1994; Trumble et d , 1995), although more isoforms are thought to exist (Kong et d , 1990; Lopez-Casillas and Kim, 1989). Bianchi et d , (1990) described a 280 KDa isoform

Figure 1.4. Model for the Domain Stmcture of ACC

The domain structure is modified from Hardie, (1989). The location of phosphorylation sites on ACC by various protein kinases is shown. The regions of sequence containing the phosphorylation sites have been illustrated in detail using the single letter amino acid code. The question mark indicates the serine residue found phosphorylated in intact adipocytes (Haystead et d , 1988) and the protein kinase that phosphorylates this site has not been identified. AMP-PK - AMP-activated protein kinase, cAMP-PK - cAMP-dependent protein kinase, modified from Davies et d ,

(1990).

bir.tin carboxvlase domain biotin domain

r --- II 1

biotin

carboxyl transferase domain

RA T 20 30 I I I I F IIG S V S E D N S E D E IS N L V K H M R SSM SG LH LV K Q G R D R K K ID SQ R Protein kinase C Casein kinase-2 C d ' * Calm odulin-dependent multiprotein kinase _{A M P-PK} cA M P-PK I I L N R M S F A S N L N H Y G M T H V A S V S D L D N A

that is distinct jfrom the 265 KDa protein and is predominantly e?q)ressed in rat cardiac and skeletal muscle. It has a higher for citrate and for acetyl-CoA than the 265 KDa form. Co-expression o f the 280 KDa form with the 265 KDa protein has been found in rat liver (Winz et al, 1994), mammary gland, brown adipose tissue (Bianchi

et d , 1990) and human liver (Abu-Elheiga et d , 1997). White adipose tissue contains

only the 265 KDa isoform (Bianchi et d , 1990). It is now known that the two isoforms of ACC are the products of separate genes (Winz et d , 1994; Widmer et d ,

1996). Immunoprécipitation studies suggest the possible co-association o f the 265 KDa and 280 KDa isoforms, therefore ACC may exist as a heterodimer as well as a homodimer (Bianchi et d , 1990). Human ACC has been characterised in HepG2 cells and molecular cloning has revealed a 264 KDa protein and evidence for another ACC- like gene which is tissue specific and could encode the 280 KDa carboxylase (Ha et d , 1994b; Abu-Elheiga et d , 1995; Widmer et d , 1996).

Citrate is an allosteric activator for virtually all acetyl-CoA carboxylases from animal cells (Moss and Lane, 1971). High concentrations o f citrate induce polymerisation of the protomeric form of the liver enzyme into a filamentous polymer composed of up to 32 protomers, disposed in an extended helical array (Gregolin et d , 1966; Lane et d , 1974; Ahmad et d , 1978). In the absence o f citrate the carboxylase protomer is virtually inactive catalytically and the polymeric filamentous form is catalytically active. Beaty and Lane, (1983a,b) demonstrated two steps which limit polymerisation of the carboxylase protomer: 1) a citrate induced conformational change which is independent of enzyme concentration and leads to an active protomeric form of the enzyme and 2) the dimérisation of the active protomer, which

constitutes the first step of polymerisation and is enzyme concentration-dependent. Dimérisation is the rate limiting step of ACC polymerisation. These polymers have been observed by electron microscopy, viscometry and ultracentrifugation o f the purified enzyme (Ahmad et d , 1978).

The mammalian isoforms 265 and 280 KDa, are predominantly present in the cytosol although there is evidence of a mitochondrial form o f ACC which is relatively inactive (Allred and Roman-Lopez, 1988). Iverson et d , (1990), Allred and Roman-Lopez, (1988) and Roman-Lopez et d , (1989) reported a sequestered form of mitochondrial ACC that accumulated during fasting (with reduced enzyme activity) and translocated to the cytosol on refeeding (associated with increased ACC activity). These authors also demonstrated mitochondrial forms of ACC in obese animals. In contrast, Moir and Zammit (1990) did not observe any such translocation of ACC from the mitochondria to the cytosol. There are several explanations for these discrepancies including, the method o f tissue sampling used, the action o f proteases and activity measurements in crude fractions relying on phosphatase activity (Moir and Zammit, 1990; Iverson et d , 1990).