Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2002-04-23
2002-11-26
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S183000, C435S232000, C435S004000, C435S015000, C548S113000, C548S303700, C424S094500
Reexamination Certificate
active
06485941
ABSTRACT:
The synthesis of fatty acids in all animals, plants, and bacteria includes a step in which acetyl-CoA is carboxylated to form malonyl-CoA. This reaction is catalyzed by acetyl-CoA carboxylase, which uses biotin as a cofactor in a two-step mechanism:
The first half-reaction is catalyzed by the biotin carboxylase component of acetyl-CoA carboxylase; this half-reaction involves the ATP-dependent carboxylation of biotin, using bicarbonate as a source of CO
2
. In the second half reaction, which is catalyzed by the carboxyltransferase component of the enzyme, the carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In both half-reactions, biotin remains covalently bound to the enzyme through an amide bond to a specific lysine residue of the biotin carboxyl carrier protein. In
Escherichia coli
, biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein are three separate proteins, while in mammalian acetyl-CoA carboxylase these three components form separate domains within a single polypeptide.
The
E. coli
form of the enzyme has served as a model system for mechanistic studies of acetyl-CoA carboxylase, because both biotin carboxylase and carboxyltransferase retain their respective activities when isolated. Moreover, biotin carboxylase and carboxyltransferase both recognize free biotin as a substrate, eliminating the need for the biotin carboxyl carrier protein during kinetic studies. Most of the recent mechanistic studies of acetyl-CoA carboxylase have focused on the biotin carboxylase component, because its gene has been cloned and overexpressed. See S. Li et al.,
J. Biol. Chem.
267, 855-863 (1992); C. Blanchard et al.,
J. Biol. Chem.
273, 19140-19145 (1998); see also C. Blanchard et al.,
Biochemistry
38, 3393-3400 (1999). The three-dimensional structure of biotin carboxylase has been determined by x-ray crystallography, as has the three-dimensional structure of biotin carboxylase complexed to ATP. See G. Waldrop et al.,
Biochemistry
33, 10249-10256 (1994); J. Thoden et al.,
J. Biol. Chem.
275, 16183-16190 (2000). Relatively little work has focused on the carboxyltransferase component, however. Carboxyltransferase is an &agr;
2
&bgr;
2
tetramer. Although the genes for the &agr; and &bgr; subunits of carboxyltransferase have been cloned, a system for the overexpression of these genes has been developed only recently. See S. Li et al.,
J. Biol. Chem.
267, 16841-16847 (1992); C. Blanchard et al.,
J. Biol. Chem.
273, 19140-19145 (1998). The overexpression system allows production of an ample supply of carboxyltransferase for structure/function studies.
Steady-state kinetic studies of recombinant carboxyltransferase have found a sequential mechanism in which both substrates must bind to the enzyme before catalysis occurs. C. Blanchard et al.,
J. Biol. Chem.
273, 19140-19145 (1998). The initial reaction rates (in the reverse direction as depicted in reaction (2) above) were consistent with an equilibrium-ordered kinetic mechanism, with malonyl-CoA binding before biotin. Note that carboxyltransferase from
E. coli
is routinely assayed in the non-physiological direction because of the availability of a facile spectrophotometric continuous assay that couples the production of acetyl-CoA with the reduction of NAD
+
by the combined reactions of citrate synthase and malate dehydrogenase. C. Blanchard et al.,
J. Biol. Chem.
273, 19140-19145 (1998); R. Guchhait et al.,
Methods Enzymol
35, 32-37 (1975). Carboxyltransferase assays also typically use biocytin in place of biotin, because biocytin gives reaction rates about three orders of magnitude higher than those for unmodified biotin. (Biocytin is biotin that is modified by attaching a lysine to the carboxyl group of the valeric acid side chain via an amide linkage with the &egr;-amino group.)
Mice lacking a gene coding for one form of acetyl-CoA carboxylase have been observed to lose weight despite increased food consumption. See L. Abu-Elheiga et al.,
Science
291, 2613-2616 (2001). Abu-Elheiga et al. and J. Thupari et al,
Biochem. Biophys. Res. Commun.
285,217-223 (2001) have suggested that mammalian acetyl-CoA carboxylase might be a potential target for anti-obesity or anti-cancer drugs, respectively.
Adipocytes are highly specialized cells that play a central role in lipid homeostasis and energy balance. Obesity, an excessive accumulation of adipose tissue, is a major risk factor in the development of Type II diabetes, cardiovascular disease, and hypertension. Recent studies have indicated that obesity and Type II diabetes may be correlated with a breakdown in the regulatory mechanisms that control adipocyte gene expression.
We have discovered a method to inhibit carboxyltransferase using bisubstrate analogs. The structure of one such bisubstrate analog (Compound 1) of carboxyltransferase is shown in
FIG. 1
, along with the substrates malonyl-CoA and biotin. Compound 1 has been synthesized, and has been shown to inhibit the carboxyltransferase component of
E. coli
acetyl-CoA carboxylase. Compound 1 also inhibits mammalian acetyl-CoA carboxylase, and thereby could act as an antiobesity agent and an anti-cancer agent.
Since Compound 1 includes the nucleotide ADP, the cell membrane is impermeable to Compound 1. However, a precursor to Compound 1, the chloroacetylated (or haloacetylated) biotin derivative Compound 2 (see FIG.
2
), is sufficiently hydrophobic to diffuse across the cell membrane. Moreover, we have shown that Compound 2 inhibits adipocyte differentiation and gene expression.
This bisubstrate analog will be useful in the treatment and prevention of obesity and diabetes.
Cancer cells also typically have high levels of fatty acid synthesis. Inhibitors of fatty acid synthesis are in clinical trials for the treatment of breast cancers. Since acetyl-CoA carboxylase—not fatty acid synthase—is the rate-limiting enzyme in fat synthesis, the inhibition of acetyl-CoA carboxylase with the bisubstrate analog could be even more effective in treating cancers than are inhibitors of fatty acid synthesis.
REFERENCES:
patent: 6242610 (2001-06-01), Strongin et al.
Abu-Elheiga, L. et al., “Continuous Fatty Oxidation and Reduced Fat Storage in Mice Lacking Acetyl-CoA Carboxylase 2,”Science 291, 2613-2616 (2001).
Amspacher et al., “Synthesis of a Reaction Intermediate Analogue of Biotin-Dependent Carboxylases via a Selective Derivatization of Biotin,” Organic Lett. 1, 99-102 (1999).
Blanchard, C. et al.,Biochemistry 38, 3393-3400 (1999).
Blanchard, C. et al., “Overexpression and Kinetic Characterization of the Carboxyltransferase Component of Acetyl-CoA Carboxylase,”J. Biol. Chem. 273, 19140-19145 (1998).
Bull, H. et al., “Mechanism-Based Inhibition of Human Steroid 5&agr;-Reductase by Finasteride; Enzyme-Catalyzed Formation of NADP-Dihydrofinasteride, a Potent Bisubstrate Analog Inhibitor,” J. Am. Chem. Soc. 118, 2359-2365 (1996).
Green, H. et al., “Spontaneous Heritable Changes Leading to Increased Adipose Conversion in 3T3 Cells,” Cell 7, 105-113 (1976).
Green, H. et al., “Sublines of Mouse 3T3 Cells That Accumulate Lipid,” Cell 1, 113-116 (1974).
Guchhait, R. et al., “Carboxyltransferase Component of Acetyl-CoA Carboxylase fromEscherichia Coli,” MethodsEnzymol 35, 32-37 (1975).
Khalil, E. et al., “Mechanism-based inhibition of the melatonin rhythm enzyme: Pharmacologic exploitation of active site functional plasticity,” Proc. Nat. Acad. Sci. USA 96, 12418-12423 (1999);.
Levert, K. et al., “A biotin analog inhibits acetyl-CoA carboxylase activity and adipogenesis,”J. Biol. Chem., vol. 277, 16347-16350 (2002).
Levert, K. et al., “A bisubstrate analog inhibitor of the carboxyltransferase component of acetyl-CoA carboxylase,”Biochem. Biophys. Res. Comm., vol. 291, pp. 1213-1217 (2002).
Li, S. et al., “The Gene Encoding the Biotin Carboxylase Subunit ofEscherichia coliAcetyl-CoA Carboxylase,”J. Biol. Chem. 267, 855-863 (1992).
Li, S. et al., “The Genes Encoding the Two Carboxyltransferase Subunits ofEscherichia coliAcetyl-CoA Carboxylase,”J. Biol. Chem. 267, 16841-1
Levert Keith L.
Stephens Jacqueline M.
Waldrop Grover L.
Board of Supervisors of Louisiana State University and Agricultu
Runnels John H.
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