Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT00572 (Asn)
 11,732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.
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PMID:Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test. 130 74

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.
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PMID:Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens. 272 36

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.
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PMID:Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups. 352 72

NADH-cytochrome b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH2-terminal region. The NH2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows: NH2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp- Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile- This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3rd Arg or 4th Ser. The NH2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols et al. (1985) J. Biol. Chem. 260, 11953-11961) differs in two amino acid assignments from our sequence.
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PMID:Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping. 365 89

Mutations in the beta subunit of Escherichia coli proton-translocating nicotinamide nucleotide transhydrogenase of the conserved residue beta Asp-213 to Asn (beta D213N) and Ile (beta D213I) resulted in the loss, respectively, of about 70% and 90% NADPH-->3-acetylpyridine adenine dinucleotide (AcPyAD) transhydrogenation and coupled proton translocation activities. However, the cyclic NADP(H)-dependent NADH-->AcPyAD transhydrogenase activities of the mutants were only approximately 35% inhibited. The latter transhydrogenation, which is not coupled to proton translocation, occurs apparently via NADP under conditions that enzyme-NADP(H) complex is stabilized. Mutations beta D213N and beta D213I also resulted in decreases in apparent KmNADPH for the NADPH-->AcPyAD and S0.5NADPH (NADPH concentration needed for half-maximal activity) for the cyclic NADH-->AcPyAD transhydrogenation reactions, and in KdNADPH, as determined by equilibrium binding studies on the purified wild-type and the beta D213I mutant enzymes. These results point to a structural role of beta Asp-213 in energy transduction and are discussed in relation to our previous suggestion that proton translocation coupled to NADPH-->NAD (or AcPyAD) transhydrogenation is driven mainly by the difference in the binding energies of NADPH and NADP.
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PMID:Proton-translocating nicotinamide nucleotide transhydrogenase of Escherichia coli. Involvement of aspartate 213 in the membrane-intercalating domain of the beta subunit in energy transduction. 762 74

The polymerase chain reaction was used to amplify six mitochondrial t-RNAs for Ala, Arg, Asn, Ser, Glu and Phe between genes for mitochondrial NADH dehydrogenases 3 and 5. With respect to Drosophila yakuba the gene order and direction of transcription is completely conserved. Analysis of secondary structure shows complete conservation of the anticodon loops but a number of differences in the dihydrouridine and T psi C loops with respect to Drosophila. However, differences are such that tertiary interactions that stabilize stacking are preserved. The use of the reported sequence in combination with PCR to explore population variability is discussed.
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PMID:Mediterranean fruit fly, Ceratitis capitata (Wiedemann), mitochondrial DNA: genes and secondary structures for six t-RNAs. 826 94

UDP-galactose 4-epimerase is one of three enzymes in the metabolic pathway that converts galactose into glucose1-phosphate. Specifically this enzyme catalyzes the interconversion of UDP-galactose and UDP-glucose. The molecular structure of the NADH/UDP-glucose abortive complex of the enzyme from Escherichia coli has been determined by X-ray diffraction analysis to a nominal resolution of 1.8 A and refined to an R-factor of 18.2% for all measurement X-ray data. The nicotinamide ring of the dinucleotide adopts the syn conformation in relationship to the ribose. Both the NADH and UDP-glucose are in the proper orientation for a B-side specific transfer from C4 of the sugar to C4 of the dinucleotide. Those residues implicated in glucose binding include Ser 124, tyr 149, Asn 179, Asn199, Arg 231, and Tyr 299. An amino acid sequence alignment of various prokaryotic and eukaryotic epimerases reveals a high degree of conservation with respect to those residues involved in both NADH and substrate binding. The nonstereospecificity displayed by epimerase was originally thought to occur through a simple rotation about the bond between the glycosyl C1 oxygen of the 4-ketose intermediate and the beta-phosphorous of the UDP moiety, thereby allowing the opposite side of the sugar to face the NADH. The present structure reveals that additional rotations about the phosphate backbone of UDP are necessary. Furthermore, the abortive complex model described here suggests that Ser 124 and Tyr 149 are likely to play important roles in the catalytic mechanism of the enzyme.
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PMID:Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism. 861 97

The Tyr residue in the mobile loop region of the soluble, domain I polypeptide (called Ths) of the proton-translocating transhydrogenase from Rhodospirillum rubrum has been substituted by Asn and by Phe. The recombinant proteins were expressed at high levels in Escherichia coli and purified to homogeneity. The two well defined resonances at 6.82 and 7.12ppm, observed in the one-dimensional proton NMR spectrum of wild-type protein, and previously attributed to the Tyr residue, were absent in both mutants. In the Tyr235 --> Phe mutant Ths, they were replaced by two new resonances at 7.26 and 7.33 ppm, characteristic of a Phe residue. In both mutants, narrow resonances attributable to Met residues (and in the Tyr235 --> Phe mutant, resonances attributable to Ala residues) were shifted relative to the wild type, but other features in the NMR spectra were unaffected. The conformational dynamics of the mobile loop closure in response to nucleotide binding by the protein were altered in the two mutants. The fluorescence emission from Trp72 was unaffected by both Tyr substitutions, and the fluorescence was still quenched by NADH. The mutant Ths proteins bound to chromatophore membranes depleted of their native Ths with undiminished affinity. In these reconstituted systems, the Km values for thio-NADP+ and NADH, during light-driven transhydrogenation, were similar to those of wild-type, but the kcat values were decreased about 2-fold. In reverse transhydrogenation, the Kmvalues for NADPH were slightly decreased in the mutants relative to wild-type, but those for acetyl pyridine adenine dinucleotide were increased about 10- and 13-fold, respectively, and the kcat values were decreased about 2- and 5-fold, respectively, in the Tyr235 --> Phe and Tyr235 --> Asn mutants. It is concluded that Tyr235 may contribute to the process of nucleotide binding and that substitution of this residue prevents proper functioning of the mobile loop in catalysis.
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PMID:Mutation of Tyr235 in the NAD(H)-binding subunit of the proton-translocating nicotinamide nucleotide transhydrogenase of Rhodospirillum rubrum affects the conformational dynamics of a mobile loop and lowers the catalytic activity of the enzyme. 862 69

Transhydrogenase from mitochondrial and bacterial membranes couples proton translocation to hydride transfer between NAD(H) and NADP(H). The enzyme has three domains, of which domains I and III protrude from the membrane. These possess the NAD(H)- and NADP(H)-binding sites, respectively, whereas domain II spans the membrane. In domain I there is a mobile loop which emanates from the surface of the protein, but which closes down upon NAD(H) binding. In this report we show that the NADP(H)-dependent reduction of acetylpyridine adenine dinucleotide by NADH catalysed by Rhodospirillum rubrum transhydrogenase has 'ping-pong' kinetics, confirming that the reaction is cyclic. We then describe the kinetic and thermodynamic properties of mutants of recombinant domain I protein from the R. rubrum enzyme, in which Tyr-235 in the mobile loop has been substituted with Phe or Asn residues (dI.Y235F and dI.Y235N, respectively). (1) Equilibrium dialysis measurements show that dI.Y235F and dI.Y235N bind NADH more weakly than wild-type domain I protein (the Kd increases twofold and fourfold, respectively). (2) Reverse transhydrogenation rates (in steady state) of domain I-depleted membrane vesicles reconstituted with either dI.Y235F or dI.Y235N are inhibited by about 50% and 78%, respectively, relative to those obtained in reconstitutions with wild-type domain I protein. (3) Reverse transhydrogenation rates (in steady state) of mixtures of recombinant domain III protein and either dI.Y235F or dI.Y235N are inhibited only by about 10% and 20%, respectively, relative to those obtained in mixtures with wild-type protein. (4) Forward transhydrogenation rates (in both the complete enzyme and in domain I:III complexes) are inhibited even less by the mutations than the reverse reactions. (5) In contrast with (1), (2) and (3), cyclic transhydrogenation was strongly inhibited in both the reconstituted membrane system and in the recombinant domain I:III complexes (only 7-8% activity remains with dI.Y235F, and only 2-3% with dI.Y235N). It was recently established that, in contrast to forward and reverse transhydrogenation, the cyclic reaction is substantially limited by the rate of hydride transfer. It is therefore concluded that mutations at Tyr-235 in the mobile loop severely disrupt the hydride transfer step in the catalytic reaction of transhydrogenase.
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PMID:Mutations at tyrosine-235 in the mobile loop region of domain I protein of transhydrogenase from Rhodospirillum rubrum strongly inhibit hydride transfer. 923 Sep 21

Cytochrome P450nor (P450nor) is a heme enzyme which catalyzes NO reduction in denitrifying fungi. Threonine 243 (Thr243) of P450nor, which corresponds to the conserved threonine of monooxygenase cytochrome P450s, was replaced by 18 different amino acids via site-directed mutagenesis. The mutation did not seriously affect the optical absorption and the CD spectral properties of the enzyme in several oxidation, ligation, or spin states or the association rate constant for association of NO with the ferric iron, suggesting subtle and local structural changes in the heme environment on Thr243 mutation. However, the NO reduction activity was dramatically altered by Thr243 mutation, depending on the properties of the replaced amino acids. The catalytic activity, as measured by N2O formation and NADH consumption, was considerably retained on substitution of Asn, Ser, and Gly for Thr243, while it was profoundly decreased or lost on substitution with other amino acids. Kinetic analysis of the reaction of the enzymes with NO and NADH indicated that the decrease in the enzymatic activity upon Thr243 mutation mainly results from a decrease in the rate of reduction of the ferric-NO complex with NADH. On the basis of these enzymatic, kinetic, and spectroscopic results, as well as on the basis of the crystal data for native P450nor [Park, S.-Y., et al. (1997) Nat. Struct. Biol. 4, 827-832], the role of the conserved threonine at the 243 position in the NO reduction reaction by P450nor is discussed. We also discuss structural similarities or differences in the vicinity of the conserved threonine between P450nor and other monooxygenase P450s.
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PMID:Site-directed mutagenesis of the conserved threonine (Thr243) of the distal helix of fungal cytochrome P450nor. 963 24


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