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)

Biliverdin reductase (molecular form 1, EC 1.3.1.24, bilirubin:NAD(P)+ oxidoreductase) carries three thiol residues. Only one of them could be alkylated when a ratio N-ethylmaleimide (NEM)/mol enzyme's SH = 90 was used. The alkylation of this thiol group inhibited the conversion of molecular form 1 to its dimer, molecular form 3; however, it did not inhibit the enzymatic activity. At a ratio of NEM/enzyme's SH = 300, two thiol residues were alkylated and the activity of the enzyme was totally inhibited. The third thiol group could not be alkylated either by NEM or by iodoacetamide. Biliverdin as well as the co-substrate NADPH protected the thiol residue essential for the enzymatic activity from alkylation. Spectroscopic evidence was obtained that this thiol group binds covalently to the C-10 of biliverdin to form a rubinoid adduct. The presence of a lysine residue, which is also essential for the enzymatic activity, could be inferred from the fact that by reduction of the Schiff base formed by the enzyme with pyridoxal phosphate the catalytic activity was irreversibly abolished. The location of a lysine residue in the vicinity of the thiol group involved in the catalytic activity was evident when the enzyme was treated with o-phthalaldehyde. The inactivation of the enzymatic activity was coincident with the formation of the fluorescent isoindole derivative which originates when the thiol and epsilon-NH2 groups are located about 3 A apart. The presence of a positively charged ammonium ion in the vicinity of the NADPH binding site was inferred from the shifts in the UVmax of NADPH from 340 nm to 327 nm and of 3-acetyl NADPH from 360 nm to 348 nm when the pyridine nucleotides bind to the reductase. The involvement of arginine residues in the enzymatic activity was established by inhibition of the latter after reaction with butanedione. This inhibition was totally protected by NADPH but not by biliverdin. The similarity of the structural features of biliverdin reductase with those of several dehydrogenases is discussed.
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PMID:Identification of the amino acid residues essential for the activity and the interconversion of the molecular forms of biliverdin reductase. 237 96

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.
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PMID:Labeling of specific lysine residues at the active site of glutamine synthetase. 241 12

The conversion of L-lysine to L-beta-lysine is catalyzed by lysine 2,3-aminomutase. The reaction involves the interchange of the 2-amino group of lysine with a hydrogen at carbon 3. As such the reaction is formally analogous to adenosylcobalamin-dependent rearrangements. However, the enzyme does not contain and is not activated by this coenzyme. Instead it contains iron and pyridoxal phosphate and is activated by S-adenosylmethionine. Earlier experiments implicated adenosyl-C-5' of S-adenosylmethionine in the hydrogen transfer mechanism, apparently in a role similar or analogous to that of adenosyl moiety of adenosylcobalamin in the B12-dependent rearrangements. The question of whether both hydrogens or only one hydrogen at adenosyl-C-5' participate in the hydrogen-transfer process has been addressed by carrying out the lysine 2,3-aminomutase reaction with S-[5'-3H] adenosylmethionine in the presence of 10 times its molar concentration of enzyme. Under these conditions all of the tritium appeared in lysine and beta-lysine, showing that C-5'-hydrogens participate. To determine whether hydrogen transfer is compulsorily intermolecular and intramolecular, various molar ratios of [3,3-2H2]lysine and unlabeled lysine were submitted to the action of lysine 2,3-aminomutase under conditions in which 10-15% conversion to beta-lysine occurred. Mass spectral analysis of the beta-lysine for monodeutero and dideutero species showed conclusively that hydrogen transfer is both intramolecular and intermolecular. The results quantitatively support our postulate that activation of the enzyme involves a transformation of S-adenosylmethionine into a form that promotes the generation of an adenosyl-5' free radical, which abstracts hydrogen from lysine to form 5'-deoxyadenosine as an intermediate.
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PMID:Lysine 2,3-aminomutase. Support for a mechanism of hydrogen transfer involving S-adenosylmethionine. 249 74

Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.
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PMID:The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230. 249 83

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) were purified to a homogeneity from rat testis. The molecular weights of aldose reductase and aldehyde reductase were estimated to be 38,000 and 41,000 by SDS-polyacrylamide gel electrophoresis, and the pI values of these enzymes were found to be 5.3 and 6.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as xylose, glucose and galactose, whereas aldehyde reductase was virtually inactive for these aldo-sugars. The Km values of aldose reductase for aldo-sugars were relatively high. When a correction was made for the fraction of aldo-sugar present as the aldehyde form, which is the real substrate of the enzyme, the Km values were much lower. Aldose reductase utilized both NADPH and NADH as coenzyme, whereas aldehyde reductase utilized only NADPH. Aldose reductase was activated significantly by sulfate ion, while aldehyde reductase was little affected. Both enzymes were inhibited strongly by the known aldose reductase inhibitors. However, aldehyde reductase was in general less susceptible to these inhibitors when compared to aldose reductase. Both aldose reductase and aldehyde reductase treated with pyridoxal 5-phosphate have lost the susceptibility to aldose reductase inhibitor, suggesting that in these two enzymes aldose reductase inhibitor interacts with a lysine residue.
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PMID:Characterization of aldose reductase and aldehyde reductase from rat testis. 250 Jan 52

A coupled assay with alpha-hydroxyglutarate dehydrogenase was used to analyze the kinetic behavior of histidinol phosphate aminotransferase from Salmonella typhymurium. Data obtained from studies of initial velocity, inhibition by products or substrate analogues, isotope exchange rates, and the determination of the equilibrium constant were consistent only with a Ping-Pong Bi Bi mechanism. Variations in inhibition patterns by different substrate analogues indicate that the microenvironment about the pyridoxal phosphate and the pyridoxamine phosphate forms of histidinol phosphate amino-transferase are different, and favor the presence of one active site with partially overlapping substrate-binding subsites for these 2 forms of the enzyme. Histidinol phosphate aminotransferase also catalyzes decomposition of beta-chloro-L-alanine to pyruvate, NH3 and Cl-; no transamination of this substrate occurs and inactivation of the enzyme accompanies this reaction. After reduction of histidinol-P aminotransferase with [3H]NaBH4, carboxymethylation, and tryptic digestion, one major radioactive peptide absorbing at 325 nm was isolated. Its primary structure was determined to be TLSK*AFALAGLR, where K* is the P-pyridoxyllysine residue. Although this peptide is only 30-40% homologous with the corresponding segment reported for other transaminases, all of these peptides are similar in placement of an hydroxyamino acid residue three residues upstream from the lysine residue, and in the cluster of hydrophobic amino acid residues immediately following the lysine residue.
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PMID:L-Histidinol phosphate aminotransferase from Salmonella typhimurium. Kinetic behavior and sequence at the pyridoxal-P binding site. 250 52

The reaction between human 4-aminobutyrate aminotransferase and the anti-epileptic drug 4-aminohex-5-enoate, an irreversible inhibitor of the enzyme, has been studied using the radiolabelled compound. The inactivated enzyme was found to lose radiolabel over a period of a few days at 37 degrees C but even in the presence of the coenzyme, pyridoxal phosphate, no enzyme activity returned. At 4 degrees C the radiolabelled inhibitor remained stably bound. The amount of enzyme-bound 4-aminohex-5-enoate was significantly less than would be expected if one mol of inhibitor was bound per mol of active site. Reversed phase chromatography of a tryptic digest of the labelled enzyme showed that, apart from material eluting at the front of the chromatogram, all of the radioactivity was in a single fraction. This fraction contained a peptide, the sequence of which indicated that it included the lysine that binds the coenzyme and that the major release of radioactivity occurred in an Edman degradation cycle corresponding to this residue.
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PMID:Stoichiometry and stability of the adduct formed between human 4-aminobutyrate aminotransferase and 4-aminohex-5-enoate: sequence of a labelled peptide. 250 53

Genes encoding the 2 subunits of tryptophan synthase in Pseudomonas putida have been identified and cloned by their similarity to the corresponding genes in Pseudomonas aeruginosa. The deduced amino acid sequences were confirmed by comparison with regions ascertained earlier by protein sequencing. The Pseudomonas amino acid sequences are 85% identical for the beta subunit and 70% identical for the alpha subunit. These sequences are compared to those of Salmonella typhimurium, where the structure is known from X-ray crystallography. Although amino acid conservation drops to 54% and 36% for the beta and alpha subunits, only 3 single residue gaps are required to maintain alignment throughout and most of the residues identified as important for catalysis or cofactor binding are conserved. The 23 residues surrounding the beta chain lysine that enters into a Schiff base linkage with the pyridoxal phosphate cofactor are compared in 13 species, including representatives from the eukaryotic and both prokaryotic kingdoms; appreciable conservation is apparent. The approximately 100 base pairs separating the trpB gene from its divergently transcribed activator gene are similar in the 2 pseudomonads, but do not resemble those of any other bacterium or fungus studied to date.
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PMID:DNA sequence of the tryptophan synthase genes of Pseudomonas putida. 250 57

1. The stability of the native conformation of the heme crevice of pyridoxal phosphate (PLP)-ferricytochromes c as assayed by the pK, for 695 nm absorption band varies considerably. The pKa values are 8.76 for cytochrome c modified by PLP at lysine 79[PLP(Lys 79)-cyt. c], 9.23 for cytochrome c modified by PLP at lysine 86 [PLP(Lys 86)-cyt.c], 9.34 for doubly PLP substituted cytochrome c at lysines 79 and 86 [(PLP)2-cyt. c], 9.50 for triply substituted cytochrome c [(PLP)3-cyt. c] and 9.06 for native cytochrome c, which indicates less stable heme crevice of PLP-cytochrome c. 2. The singly PLP-modified cytochrome c indicate decreased activities with mitochondrial cytochrome c oxidase in the following order: PLP(Lys 86)-cyt. c less than PLP(Lys 79)-cyt. c less than native cytochrome c. The high affinity Km for PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c are 0.28 microM, 0.16 microM and 0.02 microM respectively. 3. PLP-cytochromes c show decreased binding affinities to fluorescence probes 12-(9-antroyl)-stearic acid and pyrene-labelled mitoplasts. The quenching of singly PLP-modified cytochrome c depends significantly on the ionic strength.
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PMID:Interaction of pyridoxal phosphate modified cytochromes c with mitoplasts. 255 24

The potential contribution of thiolimidate formation to the increased kinetic acidity of the alpha-proton of acetyl-CoA in the carbon-carbon bond forming reaction catalyzed by 3-ketoacyl-CoA thiolase (thiolase I) from porcine heart was assessed by chemical modification and isotope exchange experiments. Thiolase is only partially inactivated after the chemical modification of lysine residues by reductive methylation, pyridoxal phosphate, or o-phthaldehyde (specific for vicinal lysine and cysteine). The thiolase-catalyzed formation of acetyl-CoA from acetoacetyl-CoA and CoASH in 18OH2 is not accompanied by the appearance of 18O in the acetyl-CoA product. These experiments effectively rule out participation of thiolimidate formation in the thiolase reaction. Other mechanisms must be employed to facilitate the abstraction of the alpha-proton of acetyl-CoA by thiolase I.
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PMID:Examination of the role of thiolimidate formation in the cleavage of acetoacetyl-CoA catalyzed by thiolase I from porcine heart. 256 19


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