Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT02079 (lysine)
 58,762 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO2.h-1.mg-1. The purified enzyme was rather stable at 37 degrees C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the Km for the substrate L-ornithine was 0.11 mM, and the Km for the cofactor pyridoxal 5-phosphate was 0.12 microM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated Ki of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a Ki of 1.6 mM and by alpha-difluoromethylornithine with a Ki of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties of purified L-ornithine decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila. 260 Aug 81

The pH dependencies of both the forward and reverse orotate phosphoribosyltransferase (ORPTase)-catalyzed reactions have been examined and determined to be dissimilar, with maximal activity for the forward reaction near to pH 8. The maximal activity of the reverse pyrophosphorolysis was observed between pH 6.5 and 7.5. Appropriate pK values were determined using computer fitting exercises. One such pK value (equal to 8.6) suggested the presence of lysine residues at the OPRTase active site. Incubations of OPRTase with the substrate analog, uracil 6-aldehyde, in the presence of sodium borohydride, suggested that this compound is a covalent modifier of OPRTase lysine residues, and substrate protection studies provided evidence that the affected lysine residues were located near to both the phosphoribosyl 1-pyrophosphate (PRibPP) and the orotate binding sites. Similar studies with pyridoxal 5-phosphate and labeled sodium borohydride as modifiers have revealed that two modified active site lysine residues per OPRTase subunit account for the loss of 90% of the enzymatic activity with this reagent. We suggest that essential lysine residues, along with divalent metal ions, are located at the OPRTase active site, and form ion-pair bonds with anionic PRibPP and orotate as these substrates bind to the enzyme. We also report that 5-azaorotate is an alternate substrate for OPRTase (Km = 75.5 +/- 0.1 microM) leading to formation of an unstable nucleotide product).
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PMID:Orotate phosphoribosyltransferase from yeast: studies of the structure of the pyrimidine substrate binding site. 266 50

A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [3H]pyridoxine 5-phosphate ([3H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [3H]PNP at 37 degrees C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The resulting Schiff's bases between PLP and AcChR amino groups were reduced with NaCNBH3, and the pyridoxylated proteins were analyzed by fluorography. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Proteinase K treatment of labeled AcChR vesicles generated a peptide of 13 kDa that could be detected with anti-PLP antibodies only when the pyridoxylation was carried out on the internal surface of the vesicles. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the alpha-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (delta) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion [Strader, C. D., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5807-5811] that is proportional to their subunit molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: application to the nicotinic acetylcholine receptor. 266 67

The nucleotide sequence of the mRNA coding for the precursor of mitochondrial serine:pyruvate aminotransferase of rat liver was determined from those of cDNA clones. The mRNA comprises at least 1533 nucleotides, except the poly(A) tail, and encodes a polypeptide consisting of 414 amino acid residues with a molecular mass of 45,834 Da. Comparison of the N-terminal amino acid sequence of mitochondrial serine:pyruvate aminotransferase with the nucleotide sequence of the mRNA showed that the mature form of the mitochondrial enzyme consisted of 390 amino acid residues of 43,210 Da. The amino acid composition of mitochondrial serine:pyruvate aminotransferase deduced from the nucleotide sequence of the cDNA showed good agreement with the composition determined on acid hydrolysis of the purified protein. The extra 24 amino acid residues correspond to the N-terminal extension peptide (pre-sequence) that is indispensable for the specific import of the precursor protein into mitochondria. In the extension peptide there are four basic amino acids distributed among hydrophobic amino acids and, as revealed on helical wheel analysis, the putative alpha-helical structure of the peptide was amphiphilic in nature. The secondary structures of the mature serine:pyruvate aminotransferase and three other aminotransferases of rat liver were predicted from their amino acid sequences. Their secondary structures exhibited a common feature and so we propose the specific lysine residue which binds pyridoxal phosphate as the active site of serine:pyruvate aminotransferase.
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PMID:Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine:pyruvate aminotransferase of rat liver. 282 18

Pyridoxal-P has been shown to be an activator of the spinach leaf ADP-glucose pyrophosphorylase. It has a higher apparent affinity than the physiological activator 3-phosphoglycerate but only activates the enzyme activity 6-fold whereas 3-phosphoglycerate gives a 25-fold activation. Reductive phosphopyridoxylation of the spinach leaf enzyme results in enzyme having less dependence on the presence of activator for activity. Labeled pyridoxal-P is incorporated into both the 54- and 51-kilodalton subunits of the spinach leaf enzyme. The incorporation is inhibited by the presence of either 3-phosphoglycerate or the allosteric inhibitor, inorganic phosphate, thus suggesting that pyridoxal phosphate is covalently bound to the allosteric activator site. The pyridoxal phosphate is bound to an epsilon-amino group of a lysine residue. The phosphopyridoxylated enzyme is more resistant to phosphate inhibition than the unmodified form. The modified 51-kDa subunit has been digested with trypsin, and the peptide containing the labeled pyridoxal phosphate has been purified via high performance liquid chromatography and sequenced. Comparison of this sequence with the deduced amino acid sequence of a rice endosperm cDNA clone indicates that the putative allosteric site of the 51-kDa subunit is close to the carboxyl-terminal. This is in contrast to what had been demonstrated for the position of the activator site of the Escherichia coli ADP-glucose pyrophosphorylase which was shown to be close to the amino-terminal of the subunit.
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PMID:Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase. 282 57

Previously, this laboratory has demonstrated [Colombo, G., & Kemp, R. G. (1976) Biochemistry 15, 1774-1780] that under appropriate conditions the citrate inhibitory binding site of rabbit skeletal muscle phosphofructokinase can be covalently modified by using pyridoxal phosphate and sodium borohydride. In the current study, phosphofructokinase was modified by [3H]pyridoxal phosphate and sodium borohydride with or without the addition of citrate to protect the ligand binding site. The modified proteins were digested with trypsin, and the peptides were separated by high-pressure liquid chromatography. A comparison of the tryptic chromatographic profiles showed that while the label was broadly distributed among nine peaks in the elution profile of the enzyme modified in the presence of the protective ligand, a single peptide contained 70% of the total radioactivity of the enzyme modified in the absence of citrate. This peptide was presumed to contain at least part of the citrate inhibitory site of the enzyme. The sequence of the peptide was determined and shown to match with positions 528-536 of phosphofructokinase with the modified residue being Lys-529. A comparison of the sequence with that of procaryotic phosphofructokinase indicated that a homologous residue in the enzyme from Bacillus stearothermophilis is critical to an allosteric site. A second peptide that was the most abundant labeled peptide in the digest of the enzyme modified in the presence of citrate was found to be identical with the second most abundant peptide of the digest from the unprotected enzyme. This peptide corresponded to residues 681-692 with the lysine at position 684 being the site of phosphopyridoxylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Amino acid sequence at the citrate allosteric site of rabbit muscle phosphofructokinase. 295 83

Tryptophan synthase, which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein that requires pyridoxal phosphate for two of its three distinct enzyme activities. Tryptophan synthase from Neurospora crassa, a homodimer of two 75-kDa subunits, was shown to bind 1 mol of pyridoxal phosphate/mol of subunit with a calculated dissociation constant for pyridoxal phosphate of 1.1 microM. The spectral properties of the holoenzyme, apoenzyme, and reconstituted holoenzyme were characterized and compared to those previously established for the heterotetrameric (alpha 2 beta 2) enzyme from Escherichia coli. The Schiff base formed between pyridoxal phosphate and the enzyme was readily reduced by sodium borohydride, but not sodium cyanoborohydride. The active site residue that binds pyridoxal phosphate, labeled by reduction of the Schiff base with tritium-labeled sodium borohydride, was determined to be lysine by high performance liquid chromatography analysis of the protein hydrolysate. A 5400-dalton peptide containing the reduced pyridoxal phosphate moiety was generated by cyanogen bromide treatment, purified and sequenced. The sequence is 85% homologous with the corresponding sequence obtained for yeast tryptophan synthase (Zalkin, H., and Yanofsky, C. (1982) J. Biol. Chem. 257, 1491-1500); the lysine derivatized by pyridoxal phosphate is located at the same relative position as that in the yeast and E. coli enzymes.
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PMID:Neurospora tryptophan synthase. Characterization of the pyridoxal phosphate binding site. 296 57

Inactivation of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase by pyridoxal 5'-P followed by reduction with NaBH4 was studied. Fructose-6-P,2-kinase is over 80% inactivated by 2 mM pyridoxal 5'-P. The stoichiometry of the pyridoxyl-P incorporation and the inactivation of the kinase follows a biphasic curve. The first P-pyridoxyl residue incorporated per protomer does not affect fructose-6-P,2-kinase, but the next two P-pyridoxyl incorporation/protomer results in 80% inactivation. The Km values for ATP and fructose-6-P of the enzymes containing varying amounts of P-pyridoxyl groups at intermediate levels of inactivation are not altered, but Vmax is decreased. Among the metabolites tested, only fructose-2,6-P2 and Mg-ATP are competitive with pyridoxal-P and protect the enzyme against the inactivation. Neither the activity nor the fructose-6-P inhibition of fructose-2,6-bisphosphatase is affected by the modification. The acid hydrolysate of the inactive P-[3H]pyridoxyl enzyme contained only [3H]pyridoxyl lysine. High performance liquid chromatography of tryptic peptides of phospho[3H]pyridoxyl enzymes reveals two peptides which were missing in the enzyme protected by fructose-2,6-P2 or ATP during the modification reaction. These peptides have been isolated, and their amino acid sequences have been determined as Asp-Gln-Asp-Lys-Tyr-Arg and Asp-Val-His-Lys-Tyr. Pyridoxal-P reacts specifically with two lysine residues at the fructose-2,6-P2-binding site of fructose-6-P,2-kinase but not that of fructose-2,6-bisphosphatase. The site may also overlap with the ATP-binding site.
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PMID:Effect of modification of lysine residues of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase with pyridoxal 5'-phosphate. 299 89

X-ray diffraction studies have been carried out using difference Fourier methods to evaluate the reaction or interaction of an affinity label and 5'-phosphate nucleotides with the gene 5 DNA binding protein in the crystalline state. In the first case the crystalline protein was reacted with pyridoxal phosphate. Pyridoxal phosphate, which has served as an affinity label for nucleotide binding sites on other enzymes, demonstrated a major site of substitution at the center of the protein's DNA binding cleft adjacent to lysine 46 as well as two other reaction sites near residues implicated in DNA binding. Difference Fourier maps of crystals exposed to 5'-dAMP, 5'-dCMP and 5'-dTMP indicated that phosphate groups were associated with most lysine and arginine side-chains on the surface of the protein but that the nucleoside portion of the ligands were generally disordered. In several cases, however, more specific binding of the nucleotides appeared to have occurred and these sites were primarily within the proposed DNA binding cleft of the protein. In particular, binding was observed near tyrosine 34, phenylalanine 73 and within the curl of the DNA binding loop containing tyrosine 26.
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PMID:X-ray studies of nucleotide binding and pyridoxal phosphate labeling of the gene 5 DNA unwinding protein. 300 44

The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha-fluoromethylhistidine is positioned at residue 322.
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PMID:Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence. 301 50


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