Publications

• Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine.

  Adesnik M., Snitkin H.

  Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybrid ... >> More

  Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5-to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism. << Less

  J Cell Physiol 95:307-317(1978) [PubMed] [EuropePMC]

• The structure and the mechanism of action of pyruvate carboxylase.

  Attwood P.V.

  Pyruvate carboxylase plays an important role in intermediary metabolism, catalysing the formation of oxaloacetate from pyruvate and HCO3-, with concomitant ATP cleavage. It thus provides oxaloacetate for gluconeogenesis and replenishing tricarboxylic acid cycle intermediates for fatty acid, amino ... >> More

  Pyruvate carboxylase plays an important role in intermediary metabolism, catalysing the formation of oxaloacetate from pyruvate and HCO3-, with concomitant ATP cleavage. It thus provides oxaloacetate for gluconeogenesis and replenishing tricarboxylic acid cycle intermediates for fatty acid, amino acid and neurotransmitter synthesis. The enzyme is highly conserved and is found in a great variety of organisms including fungi, bacteria and plants as well as higher organisms. It is a member of a group of biotin-dependent enzymes and the biotin prosthetic group is covalently bound to the polypeptide chain of the enzyme, there normally being four such chains in the native, tetrameric enzyme. The overall reaction catalysed by pyruvate carboxylase involves two partial reactions that occur at spatially separate subsites within the active site, with the covalently bound biotin acting as a mobile carboxyl group carrier. In the first partial reaction, biotin is carboxylated using ATP and HCO3-as substrates whilst in the second partial reaction, the carboxyl group from carboxybiotin is transferred to pyruvate. The chemical mechanisms of the partial reactions and some of the roles played by amino acid residues of the enzyme in catalysing the reaction have been elucidated. The domain structure of the yeast enzyme has been deduced by comparing its amino acid sequence with those of enzymes that have similar catalytic functions. The quaternary structures of the pyruvate carboxylases studied so far, all involve a tetrahedron-like arrangement of the subunits. The major regulator of enzyme activity, acetyl CoA, stimulates the cleavage of ATP in the first partial reaction and in addition it has been shown to induce a conformational change in the tetrameric structure of the enzyme. In the past, the lack of any detailed structural information on the enzyme has hampered efforts to fully understand how this and other biotin-dependent enzymes function and are regulated. With the recent cloning of the enzyme from a variety of sources and the performance of three-dimensional structural studies, the next few years should see much progress in our understanding the mechanism of action of this enzyme. << Less

  Int J Biochem Cell Biol 27:231-249(1995) [PubMed] [EuropePMC]

• Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme.

  Adina-Zada A., Jitrapakdee S., Surinya K.H., McIldowie M.J., Piggott M.J., Cleland W.W., Wallace J.C., Attwood P.V.

  Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillu ... >> More

  Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the K(m)s for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5'-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful. << Less

  Int J Biochem Cell Biol 40:1743-1752(2008) [PubMed] [EuropePMC]

• Structure, mechanism and regulation of pyruvate carboxylase.

  Jitrapakdee S., St Maurice M., Rayment I., Cleland W.W., Wallace J.C., Attwood P.V.

  PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO(3)(-)- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochem ... >> More

  PC (pyruvate carboxylase) is a biotin-containing enzyme that catalyses the HCO(3)(-)- and MgATP-dependent carboxylation of pyruvate to form oxaloacetate. This is a very important anaplerotic reaction, replenishing oxaloacetate withdrawn from the tricarboxylic acid cycle for various pivotal biochemical pathways. PC is therefore considered as an enzyme that is crucial for intermediary metabolism, controlling fuel partitioning toward gluconeogenesis or lipogenesis and in insulin secretion. The enzyme was discovered in 1959 and over the last decade there has been much progress in understanding its structure and function. PC from most organisms is a tetrameric protein that is allosterically regulated by acetyl-CoA and aspartate. High-resolution crystal structures of the holoenzyme with various ligands bound have recently been determined, and have revealed details of the binding sites and the relative positions of the biotin carboxylase, carboxyltransferase and biotin carboxyl carrier domains, and also a unique allosteric effector domain. In the presence of the allosteric effector, acetyl-CoA, the biotin moiety transfers the carboxy group between the biotin carboxylase domain active site on one polypeptide chain and the carboxyltransferase active site on the adjacent antiparallel polypeptide chain. In addition, the bona fide role of PC in the non-gluconeogenic tissues has been studied using a combination of classical biochemistry and genetic approaches. The first cloning of the promoter of the PC gene in mammals and subsequent transcriptional studies reveal some key cognate transcription factors regulating tissue-specific expression. The present review summarizes these advances and also offers some prospects in terms of future directions for the study of this important enzyme. << Less

  Biochem J 413:369-387(2008) [PubMed] [EuropePMC]

• Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli.

  Zeczycki T.N., St Maurice M., Jitrapakdee S., Wallace J.C., Attwood P.V., Cleland W.W.

  The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of ... >> More

  The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO(2) to form oxaloacetate and complete the reaction. << Less

  Biochemistry 48:4305-4313(2009) [PubMed] [EuropePMC]