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Query: EC:6.4.1.1 (pyruvate carboxylase)
 1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid method for the purification of pyruvate carboxylase from rat liver has been developed. The method involves extraction of the enzyme from frozen liver powder followed by polyethylene glycol fractionation and avidin-affinity chromatography. The purified enzyme has a specific activity of 9-10 mumol/min/mg protein when assayed at 22 degrees C in the presence of acetyl-CoA. Polyacrylamide gel electrophoresis of the preparation in the presence of sodium dodecyl sulfate showed the presence of one protein band with an estimated Mr 125,000 and no significant contamination by other biotin-containing enzymes. In addition to being rapid, the method is advantageous because prior isolation of mitochondria is not necessary. Using these preparations we have determined the sequence of the first 15 amino acids from the NH2-terminal end of the molecule to be Ser-Gly-Pro-Val-Ala-Pro-Leu-Asn-Val-Leu-Leu-Leu-Glu-Tyr-Pro. The sequence of the 24 amino acid residues around the biotin site was determined to be Gly-Ala-Pro-Leu-Val-Leu-Ser-Ala-Met-biocytin-Met-Glu-Thr-Val-Val-Thr-Ser -Pro- Thr-Glu-Gly-Thr-Ile-Arg.
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PMID:A rapid purification method for rat liver pyruvate carboxylase and amino acid sequence analyses of NH2-terminal and biotin peptide. 317 28

Biotin-dependent carboxylases require covalently bound biotin for enzymatic activity. The biotin is attached through a lysine residue, which in a number of bacterial, avian, and mammalian carboxylases, is found within the conserved sequence Ala-Met-Lys-Met. We have determined the partial nucleotide sequence of cDNA clones for human propionyl-CoA carboxylase and pyruvate carboxylase. The predicted amino acid sequence of both these proteins contains the conserved tetrapeptide 35 residues from the carboxy terminus. In addition, both proteins contain the tripeptide, Pro-Met-Pro, 26 residues toward the amino terminus from the biotin attachment site. The overall amino acid homology through this region is 43%. Similar findings have been made for the biotin-containing polypeptides of transcarboxylase of Propionibacterium shermanii and acetyl-CoA carboxylase of Escherichia coli (W. L. Maloy, B. U. Bowien, G. K. Zwolinski, K. G. Kumar, and H. G. Wood (1979) J. Biol. Chem. 254, 11615-11622). The implications of this sequence conservation with regard to the function and evolution of biotin-dependent carboxylases is discussed. We propose that the 60 amino acids surrounding the biotin site are bounded by a proline "hinge" and the carboxy terminus has remained conserved as a result of constraints imposed by biotinylation of the enzyme.
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PMID:Sequence homology around the biotin-binding site of human propionyl-CoA carboxylase and pyruvate carboxylase. 355 48

An oligonucleotide probe specific for the amino acid sequence at the biotin site in pyruvate carboxylase was used to screen a human liver cDNA library. Nine cDNA clones were isolated and three proved to be pyruvate carboxylase clones based on nucleotide sequencing and Northern blotting. The biotin site amino acid sequence of human pyruvate carboxylase agreed perfectly with that of the sheep enzyme in 14 consecutive positions. The highly conserved amino acid sequence, Ala-Met-Lys-Met, found at the biotin site in most biotin-containing carboxylases was also present in human pyruvate carboxylase. The termination codon was located 35 residues 3' to the lysine residue at which the biotin is attached. Therefore, the biotin cofactor is covalently linked near the carboxyl-terminal end of the carboxylase protein. These data are consistent with that observed for other biotin-containing carboxylases and strongly suggests that the genes encoding the biotin-containing carboxylases may have evolved from a common ancestral gene. Northern blotting of mRNA isolated from human, baboon, and rat liver demonstrated that the pyruvate carboxylase mRNA was 4.2 kilobase pairs in length in all species examined. Southern blot analysis of genomic DNA isolated from human-Chinese hamster somatic cell hybrids localized the pyruvate carboxylase gene on the long arm of human chromosome 11. The human cDNA was also used to quantitate pyruvate carboxylase mRNA levels in a differentiating mouse preadipocyte cell line. These data demonstrated that pyruvate carboxylase mRNA content increased 23-fold in 7 days after the onset of differentiation.
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PMID:Molecular cloning of a cDNA for human pyruvate carboxylase. Structural relationship to other biotin-containing carboxylases and regulation of mRNA content in differentiating preadipocytes. 654 74

Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.
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PMID:Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains. 1104 65