Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme (original) (raw)
Related papers
Biochemistry, 1992
When chicken liver pyruvate carboxylase was incubated with either H14C03-or y-[32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. Thecomplex was catalytically competent, as determined by its subsequent ability to transfer either 14C02 to pyruvate or 32P to ADP. While the carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [ 14C]carboxyphospho+nzyme and the~arboxy[~~P]phosphoenzyme had similar half-lives. Acetyl-coA was shown to be involved in the conversion of the carboxyphosphoenzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-coA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme.
Site-directed Mutagenesis of ATP Binding Residues of Biotin Carboxylase
Journal of Biological Chemistry, 2001
Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are D-Ala D-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase, N 5-carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for sitedirected mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, the K m value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30-and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.
Movement of the Biotin Carboxylase B-domain as a Result of ATP Binding
Journal of Biological Chemistry, 2000
Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In Escherichia coli, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. The biotin carboxylase component has served for many years as a paradigm for mechanistic studies devoted toward understanding more complicated biotin-dependent carboxylases. The three-dimensional x-ray structure of an unliganded form of E. coli biotin carboxylase was originally solved in 1994 to 2.4-Å resolution. This study revealed the architecture of the enzyme and demonstrated that the protein belongs to the ATP-grasp superfamily. Here we describe the threedimensional structure of the E. coli biotin carboxylase complexed with ATP and determined to 2.5-Å resolution. The major conformational change that occurs upon nucleotide binding is a rotation of approximately 45 o of one domain relative to the other domains thereby closing off the active site pocket. Key residues involved in binding the nucleotide to the protein include Lys-116, His-236, and Glu-201. The backbone amide groups of Gly-165 and Gly-166 participate in hydrogen bonding interactions with the phosphoryl oxygens of the nucleotide. A comparison of this closed form of biotin carboxylase with carbamoyl-phosphate synthetase is presented.
Construction of new forms of pyruvate carboxylase to assess the allosteric regulation by acetyl-CoA
Protein Engineering Design and Selection, 2005
The single polypeptide chain of Bacillus thermodenitrificans pyruvate carboxylase (PC) is composed of the biotin carboxylase (BC), carboxyl transferase (CT) and biotin carboxyl carrier protein (BCCP) domains from the amino terminus. This polypeptide chain was divided into two between the CT and BCCP domains. The resulting proteins, PC-(BC + CT) and PC-(BCCP), were expressed in Escherichia coli separately, purified to homogeneity and characterized. PC-(BC + CT) was 4% as active as native PC in the carboxylation of pyruvate with PC-(BCCP) as substrate with a K m of 39 mM. Moreover, acetyl-CoA stimulated the carboxylation of PC-(BCCP) about 3-fold, whereas it was without effect in the corresponding reaction with free biotin. In addition to these engineered proteins, another form of enzyme was also constructed in which the BC domain of B.thermodenitrificans PC was replaced with the BC subunit of Aquifex aeolicus PC, whose activity is independent of acetyl-CoA. The resulting chimera was about 7% as active as native PC, but its activity was independent of acetyl-CoA. On the basis of these observations, the mechanism by which acetyl-CoA regulates the reaction of PC is discussed.
The Biochemical journal, 1981
The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypy...
Biochimie, 2008
The stereochemistry of CO 2 addition to phosphoenolpyruvate (PEP) to yield oxaloacetate catalyzed by ATP-dependent Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens PEP carboxykinases was determined using (Z)-3-fluorophosphoenolpyruvate ((Z)-F-PEP) as a substrate analog. A. succiniciproducens and S. cerevisiae PEP carboxykinases utilized (Z)-F-PEP with 1/14 and 1/47 the respective K m values for PEP. On the other hand, in the bacterial and yeast enzymes k cat was reduced to 1/67 and 1/48 the value with PEP, respectively. The binding affinity of pyridoxylphosphate-labeled S. cerevisiae and A. succiniciproducens PEP carboxykinases for PEP and (Z)-F-PEP was checked and found to be of similar magnitude for both substrates, suggesting that the lowered K m values for the fluorine-containing PEP analog are due to kinetic effects. The lowered k cat values when using (Z)-F-PEP as substrate suggest that the electron withdrawing effect of fluorine affects the nucleophilic attack of the double bond of (Z)-F-PEP to CO 2 . For the stereochemical analyses, the carboxylation of (Z)-F-PEP was coupled to malate dehydrogenase to yield 3-fluoromalate, which was analyzed by 19 F NMR. The fluoromalate obtained was identified as (2R, 3R)-3fluoromalate for both the A. succiniciproducens and S. cerevisiae PEP carboxykinases, thus indicating that CO 2 addition to (Z)-F-PEP, and hence PEP, takes place through the 2-si face of the double bond. These results, together with previously published data [Rose, I.A. et al. J. Biol. Chem. 244 (1969) 6130e6133; Hwang, S.H. and Nowak, T. Biochemistry 25 (1986) 5590e5595] indicate that PEP carboxykinases, no matter their nucleotide specificity, catalyze the carboxylation of PEP from the 2-si face of the double bond.
Biochemistry, 1996
Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5′-triphosphate (ATP), orthophosphate (P i ), and pyruvate with adenosine 5′-monophosphate (AMP), pyrophosphate (PP i ), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [ 32 P]-8-azidoadenosine 5′-triphosphate ([ 32 P]-8-azidoATP). Subtilisin cleavage of the [R-32 P]-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues 1 and 226 and the other between residues 227 and 334. Saturation of the ATP binding site with adenylyl imidodiphosphate afforded protection against photolabeling. Next, small peptide fragments of [γ-32 P]-8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or R-chymotrypsin. A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTIEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in R-chymotrypsin-treated PPDK), thus locating one of the positions of covalent modification. Next, catalysis by site-directed mutants generated by amino acid replacement of invariant residues of the PPDK N-terminal domain was tested. K163L, D168A, D170A, D175A, K177L, and G248I PPDK mutants retained substantial catalytic activity while G254I, R337L, and E323L PPDK mutants were inhibited. Comparison of the steady-state kinetic constants measured (at pH 6.8, 25°C) for wild-type PPDK (k cat ) 36 s -1 , AMP K m ) 7 µM, PP i K m ) 70 µM, PEP K m ) 27 µM) to those of R337L PPDK (k cat ) 2 s -1 , AMP K m ) 85 µM, PP i K m ) 3700 µM, PEP K m ) 6 µM) and G254I PPDK (k cat ) 0.1 s -1 , AMP K m ) 1300 µM, PP i K m ) 1200 µM, PEP K m ) 12 µM) indicated impaired catalysis of the nucleotide partial reaction (E‚ATP‚P i f E-PP‚AMP‚P i f E-P‚AMP‚PP i ) in these mutants. The single turnover reactions of [ 32 P]PEP to [ 32 P]E-P‚pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [ 32 P]E-PP/[ 32 P]E-P in single turnover reactions of [ -32 P]ATP/P i was significantly inhibited. Finally, the location of the adenosine 5′-diphosphate binding site within the nucleotide binding domain of D-alanine-D-alanine ligase, a structural homologue of the PPDK N-terminal domain ) Proc. Natl. Acad. Sci. U.S.A. 93, 2652-2657 indicates, by analogy, the location of the nucleotide binding site in PPDK. Residues G254, R337, and E323 as well as the site of photoaffinity labeling are located within this region.
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1992
sulfo-l.naphthyl)ethylenediamine, incorporated 0.95 mol of the fluorescent moiety per moi of enzyme subunit. Reagent incorporation was completely protected by the presence of ADP plus MnCI 2. The labeled protein was digested with trypsin after carboxymethylation. Two labeled peptides were isolated by reverse-phase high-performance liquid chromatography and were sequenced by gas-phase automatic Edman degradation. Both peptides contained overlapping amino acid sequences from .Asn-358 to Lys-375, thus identifying Cys-364 as the reactive amino acid residue. The position of the target amino acid residue is immediately preceding a putative phosphorylbinding sequence proposed for some nucleotide-binding proteins.
Journal of Molecular Biology, 2010
Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 Å resolution and containing bound MgADP, and the other at 2.1 Å resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K m CP and k cat triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigidbody movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/ acetate and bicarbonate, rendering CK highly selective for CP/carbamate.