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)

Pyridoxal phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was inactivated by (S)-alpha-fluoromethylhistidine by a pseudo first-order reaction, with KI and k inact values of 0.1 mM and 32.2 min-1, respectively, and was most efficient at pH 6.5-7.0. Both L-histidine and the competitive inhibitor, L-histidine methyl ester, protected against inactivation. The apoenzyme was not inactivated. These findings indicate that inhibition is a mechanism-based process. Under optimal conditions a single molecule of alpha-fluoromethylhistidine inactivates one enzyme subunit, indicating that no escaping side reaction occurs during the inactivation process. The bound inactivator is not released by dialysis of the native protein but is released upon denaturation by heat or urea. This released product was not fully characterized, but it contains the tritium of ring-labeled alpha-fluoromethyl-[3H]histidine, exhibits the spectral properties of a 3-hydroxypyridine derivative, and does not yield any amino acids on hydrolysis. The label was much more stable following borohydride reduction of the inactivated protein, and a tryptic peptide containing the modified residue was isolated. Sequencing of this peptide and the corresponding peptide from the native enzyme revealed that the inactivator binds to a serine residue of the holoenzyme. Two P-pyridoxyl peptides from tryptic or CNBr digests of the NaBH4-reduced enzyme were also isolated. Sequence and compositional data obtained with these peptides showed that the serine residue to which the inhibitor binds is not near the lysine residue that binds pyridoxal-P in the primary sequence of the protein, although the two residues must be near one another in the three-dimensional structure to account for these results. A speculative mechanism for inactivation, consistent with the experimental findings, is presented.
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PMID:Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Inactivation by alpha-fluoromethylhistidine and comparative sequences at the inhibitor- and coenzyme-binding sites. 373 45

Activated ribulosebisphosphate carboxylase/oxygenase from spinach was treated with glyoxylate plus or minus the transition-state analog, carboxyarabinitol bisphosphate, or the inactive enzyme with pyridoxal phosphate plus or minus the substrate, ribulose bisphosphate. Covalently modified adducts with glyoxylate or pyridoxal phosphate were formed following reduction with sodium borohydride. The derivatized enzymes were carboxymethylated and digested with trypsin; the labeled peptides which were unique to the unprotected samples were purified by ion-exchange chromatography and gel filtration. Both glyoxylate and pyridoxal phosphate were associated with only one major peptide, which in each case was subjected to amino acid analysis and sequencing. The sequence was -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys(Cm)-Thr-Ile-Lys-Lys*-Pro-Lys-, with both reagents exhibiting specificity for the same lysine residue as indicated by the asterisk. This peptide is identical to that previously isolated from spinach carboxylase labeled with either of two different phosphorylated affinity reagents and homologous to one from Rhodospirillum rubrum carboxylase modified by pyridoxal phosphate. The species invariance of this lysine residue, number 175, and the substantial conservation of adjacent sequence support the probability for a functional role in catalysis of the lysyl epsilon-amino group.
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PMID:Characterization of an active-site peptide modified by glyoxylate and pyridoxal phosphate from spinach ribulosebisphosphate carboxylase/oxygenase. 386 Jan 89

Crystallographic studies of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) in conjunction with chemical modification experiments have led to the suggestion that the active sites of the enzyme are at the interfaces between adjacent polypeptide chains of the catalytic trimers and involve joint participation of amino acid residues from the adjoining chains. However, the precise locations of the active sites and of the residues involved in catalysis are not known. To test the hypothesis that the active sites are shared between chains, we constructed hybrid trimers in which two chains were modified at one presumed active site residue and the third chain was altered at a different active site residue. One parental trimer was a reduced pyridoxal phosphate derivative in which lysine-84 was modified and the other was a mutant protein in which tyrosine-165 was converted to serine by site-directed mutagenesis. Incubating mixtures of these two virtually inactive derivatives under conditions promoting interchain exchange led to a large increase in enzyme activity corresponding approximately to the formation of one active site per trimer. The purified hybrid trimers, containing either two pyridoxylated and one mutant chain or vice versa, had 23% and 28%, respectively, the activity of native wild-type catalytic trimers, compared to 5% and 3% for the parental trimers. The most likely explanation for this large increase in activity is the formation of one "native" active site in each of the hybrid trimers. The results constitute strong evidence for shared active sites in aspartate transcarbamoylase.
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PMID:Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase. 388 63

The gene for aspartate aminotransferase from E. coli (aspC) was subcloned into M13 phage and sequenced using the Sanger dideoxy method with synthetic oligonucleotide primers. A mutant gene was constructed using site-directed mutagenesis techniques in which the codon for the lysine that forms the Schiffs base with pyridoxal phosphate was replaced with one coding for alanine. The mutant gene was expressed under control of the Tac promoter to overproduce a mutant protein lacking enzymatic activity.
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PMID:Site-directed mutagenesis of aspartate aminotransferase from E. coli. 390 32

The stereochemical course of the wheat germ meso-diaminopimelate (DAP) decarboxylase reaction is compared to that of the decarboxylase isolated from Bacillus sphaericus, which has been reported to proceed with an unusual inversion of configuration [Asada, Y., Tanizawa, K., Sawada, S., Suzuki, T., Misono, H., & Soda, K. (1981) Biochemistry 20, 6881-6886]. Reaction of each enzyme with either unlabeled diaminopimelic acid in D2O or [2,6-2H2]diaminopimelic acid in H2O gave stereospecifically deuterium-labeled lysine samples that were derivatized with (-)-camphanoyl chloride and diazomethane. Analysis by two-dimensional 1H-13C heteronuclear NMR shift correlation spectroscopy with 2H decoupling confirmed the stereochemistry of the B. sphaericus enzyme reaction and showed that the eukaryotic wheat germ meso-DAP decarboxylase also operates with inversion of configuration. This suggests similar mechanisms for the prokaryotic and eukaryotic enzymes and contrasts the retention mode observed with other pyridoxal phosphate dependent alpha-decarboxylases.
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PMID:Stereochemistry of lysine formation by meso-diaminopimelate decarboxylase from wheat germ: use of 1H-13C NMR shift correlation to detect stereospecific deuterium labeling. 392 76

The spatial structure of cytosolic chicken aspartate aminotransferase (AAT) has been determined by X-ray crystallographic analysis at 2.8 A resolution. AAT consists of two chemically identical subunits. Each subunit can be subdivided into the large pyridoxal phosphate (PLP) binding domain and the small domain. The two active sites of AAT are situated in deep clefts at the subunit interface. The binding of PLP and 2-oxoglutarate is described. Conformations of the following enzyme forms have been compared by difference Fourier syntheses: the nonliganded PLP-form in phosphate and acetate buffers; the non-liganded pyridoxamine phosphate (PMP) form; complexes of the PLP-form with glutarate and 2-oxoglutarate. Lattice-induced dynamic asymmetry of the dimeric AAT molecules was revealed. In one subunit the small domain is mobile and shifted either toward the active site ("closed" conformation) or in the opposite direction ("open" conformation). The closed conformation is induced by the binding of dicarboxylate anions. In the second subunit the small domain is immobile and shifted toward the active site in all enzyme forms or complexes studied. In this subunit, there occurs a rotation of the PLP ring by approximately 20 degrees toward the substrate site. The rotation is observed when crystals are soaked in 0.6 saturated (NH4)2SO4 solution buffered with 0.3 M potassium phosphate, pH 7.5; it was explained by formation of an external aldimine between PLP and NH3. This aldimine is not formed in the presence of dicarboxylates or acetate. It was inferred that dicarboxylate or acetate anions stabilize the internal PLP-lysine aldimine and prevent its reaction with ammonia. Conversion of AAT from the PLP- to PMP-form is accompanied by rotation of the coenzyme ring by approximately 20 degrees; the rotation occurs in both subunits.
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PMID:[Cytosol aspartate aminotransferase from the chicken heart: three-dimensional structure at 2.8 angstroms resolution and the characteristic conformation of various enzyme forms]. 398 8

A pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was purified to homogeneity. The enzyme is a tetramer (Mr 170,000) of identical subunits and binds 4 pyridoxal-P/tetramer; it is resolved by dialysis against cysteine at pH 6.8. Between pH 6.2 and 8.8, the holoenzyme shows pH-independent absorbance maxima at 333 and 416 nm. Vmax/Km is highest at pH 6.5; this optimum reflects chiefly increased Km values for histidine at lower or higher pH values, whereas Vmax is highest at pH 5.0 and decreases only moderately between pH 5.0 and 8.0. The enzyme also decarboxylates beta-(2-pyridyl)alanine and N tau-methylhistidine (but not N pi-methylhistidine); arginine, lysine, and ornithine are neither substrates nor inhibitors. The hydrazine analogue of histidine, 2-hydrazino-3-(4-imidazolyl)propionic acid, is a very potent competitive inhibitor; other carbonyl reagents and a variety of carboxyl- or amino-substituted histidines also inhibit competitively. alpha-Fluoromethylhistidine is a potent irreversible inhibitor of the enzyme; alpha-methylhistidine is a competitive inhibitor/substrate that is decarboxylated slowly and undergoes a slow decarboxylation-dependent transamination that converts the holoenzyme to pyridoxamine-P and apoenzyme. Dithiothreitol and other simple thiols are mixed-type inhibitors that interact with pyridoxal-P at the active site to form complexes (lambda max congruent to 340 nm), presumably the corresponding thioalkylamines, without resolving the holoenzyme. This histidine decarboxylase (Vmax = 72 mumol X min-1 X mg-1) is much more active than "homogeneous" preparations of mammalian pyridoxal-P-dependent histidine decarboxylase (Vmax congruent to 1.0) and is about equal in activity to the pyruvoyl-dependent histidine decarboxylases from Gram-positive bacteria.
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PMID:Purification and properties of a pyridoxal 5'-phosphate-dependent histidine decarboxylase from Morganella morganii AM-15. 399 48

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and L-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5'-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.
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PMID:Mitochondrial nicotinamide nucleotide transhydrogenase: inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate. 406 2

Cytosolic chicken heart aspartate aminotransferase (EC 2.6.1.1) was incorporated in polyacrylamide gel and partially oriented by compressing the gel block in two mutually perpendicular directions. The linear dichroism (LD) was recorded in a dichrograph equipped with a quarter-wavelength device which transforms circularly polarized light into linearly polarized. Spectra were resolved with lognormal distribution curves. A marked difference has been found between reduced linear dichroism values (LD/A) in the absorption bands of the protonated (430 nm) and nonprotonated (360 nm) forms of the internal pyridoxal phosphate--lysine aldimine. This finding indicates that protonation of the internal aldimine bond induces a change in direction of the transition dipole moment within the coenzyme ring or reorientation of the ring. Formation of the external aldimine with 2-methylaspartate is accompanied by a decrease of the reduced LD value in the 430 nm band. On the other hand, binding of the dicarboxylate anions, which imitates formation of the noncovalent adsorption Michaelis complex, results in a marked increase of the reduced LD value in the 430 nm band. These data suggest that the coenzyme ring tilts in opposite directions upon noncovalent substrate binding and upon subsequent formation of the external aldimine.
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PMID:[Linear dichroism of chicken cytosol aspartate aminotransferase oriented in polyacrylamide gel]. 407 36

Lathyrogens decrease collagen and elastin cross-linking by inhibiting lysine oxidase. The lathyrogens isoniazid and semicarbazide decrease liver pyridoxal phosphate and are teratogenic; all their effects are reversed by pyridoxal. beta-Aminopropionitrile, another lathyrogen, does not affect liver pyridoxal phosphate, and its lathyrogenic and teratogenic effects are not reversed by pyridoxal. Time courses of these effects differ greatly, suggesting enzyme inhibition by different mechanisms.
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PMID:The inhibition of protein-lysine 6-oxidase by various lathyrogens. Evidence for two different mechanisms. 408 35


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