scholarly journals Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1

1997 ◽  
Vol 328 (1) ◽  
pp. 193-197 ◽  
Author(s):  
Stephanie WRAGG ◽  
K. Fred HAGEN ◽  
A. Lawrence TABAK
Keyword(s):  
Recombinant Proteins ◽  
Initial Rate ◽  
Initial Step ◽  
Enzymic Activity ◽  
Histidine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Histidine Residues ◽  
Sds Page ◽  
Enzyme Biosynthesis

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) catalyse the initial step of mucin-type O-glycosylation. The activity of bovine ppGaNTase-T1 isoenzyme was inhibited by diethyl pyrocarbonate (DEPC) modification. Activity was partially restored by hydroxylamine treatment, indicating that one of the reactive residues was a histidine. The transferase was protected against DEPC inactivation when UDP-GalNAc and EPO-G, a peptide pseudo-substrate PPDAAGAAPLR, were simultaneously present, while presence of EPO-G alone did not alter DEPC inactivation. However, inclusion of UDP-GalNAc alone potentiated DEPC-inhibition of the enzyme, suggesting that UDP-GalNAc binding changes the accessibility or reactivity of an essential histidine residue. Deletion of the first 56 amino acids (including one hisitidine residue) yielded a fully active secreted form of the bovine ppGaNTase-T1 enzyme. Each of the 14 remaining histidines in the enzyme were mutated to alanine, and the recombinant mutants were recovered from COS7 cells. The mutation of histidine residues His211 → Ala and His344 → Ala resulted in recombinant proteins with no detectable enzymic activity. A significant decrease in the initial rate of GalNAc transfer to the substrate was observed with mutants His125 → Ala and His341 → Ala (1% and 6% of wild-type activity respectively). Mutation of the remaining ten histidine residues yielded mutants that were indistinguishable from the wild-type enzyme. Mutagenesis and SDS/PAGE analysis of all N-glycosylation sequons revealed that positions N-95 and N-552 are occupied by N-linked sugars in COS7 cells. Ablation of either site did not perturb enzyme biosynthesis or enzyme activity.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

scholarly journals Kinetic alteration of a human dihydrodiol/3α-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine

2000 ◽  
Vol 352 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Tatuya OHTA ◽  
Syuhei ISHIKURA ◽  
Syunichi SHINTANI ◽  
Noriyuki USAMI ◽  
Akira HARA
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Clofibric Acid ◽  
The Other ◽  
Flufenamic Acid ◽  
Histidine Residue ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Competitive Inhibitors ◽  
Dihydrodiol Dehydrogenase

Human dihydrodiol dehydrogenase with 3α-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1Ő1C4) that belong to the aldoŐketo reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1Ő1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the Km for NADP+ and differently influenced the Km and kcat for substrates depending on their structures. The kinetic constants for bile acids with a 12α-hydroxy group were decreased 1.5Ő7-fold and those for the other substrates were increased 1.3Ő9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17β-oestradiol, phenolphthalein and flufenamic acid and 3,5,3´,5´-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the Km for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators.

scholarly journals Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase

1997 ◽  
Vol 326 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Shoshana KEYNAN ◽  
Nigel M. HOOPER ◽  
Anthony J. TURNER
Keyword(s):  
Specific Activity ◽  
Mammalian Species ◽  
Competitive Inhibitor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Renal Metabolism ◽  
Leukotriene D4 ◽  
Histidine Residues

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

scholarly journals Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme

1990 ◽  
Vol 270 (2) ◽  
pp. 319-323 ◽  
Author(s):  
A K Drabikowska ◽  
G Woźniak
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Active Site ◽  
Order Rate Constant ◽  
Enzymic Activity ◽  
Histidine Residue ◽  
Uridine Phosphorylase ◽  
High Concentration ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.

scholarly journals Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily

1999 ◽  
Vol 344 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Melissa BOWKER-KINLEY ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Catalytic Domain ◽  
Heat Shock Protein 90 ◽  
Enzymic Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
C Terminus ◽  
Km Value

In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent Vmax values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent Km value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 μM). In contrast, Gly-284Ala had only a slightly higher Km value than the wild-type enzyme (28 versus 16 μM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The Kd value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 μM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].

scholarly journals Histidine-193 of rat glucosylceramide synthase resides in a UDP-glucose- and inhibitor (D-threo-1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol)-binding region: a biochemical and mutational study

1999 ◽  
Vol 341 (2) ◽  
pp. 395-400 ◽  
Author(s):  
Kangjian WU ◽  
David L. MARKS ◽  
Rikio WATANABE ◽  
Pascal PAUL ◽  
Niveda RAJAN ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Mechanism ◽  
Chemotherapeutic Agents ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glucosylceramide Synthase ◽  
Mutant Proteins ◽  
Histidine Residues ◽  
Common Precursor ◽  
Pharmaceutical Properties

Glucosylceramide synthase (GCS) catalyses the transfer of glucose from UDP-glucose (UDP-Glc) to ceramide to form glucosylceramide, the common precursor of most higher-order glycosphingolipids. Inhibition of GCS activity has been proposed as a possible target of chemotherapeutic agents for a number of diseases, including cancer. Design of new GCS inhibitors with desirable pharmaceutical properties is hampered by lack of knowledge of the secondary structure or catalytic mechanism of the GCS protein. Thus we cloned the rat homologue of GCS to begin studies to identify its catalytic regions. The histidine-modifying agent diethyl pyrocarbonate (DEPC) inhibited recombinant rat GCS expressed in bacteria; this inhibition was rapidly reversible by hydroxylamine and could be diminished by preincubation of GCS with UDP-Glc. These data suggest that DEPC acts on histidine residues within or near the UDP-Glc-binding site of GCS. Mutant proteins were expressed in which the eight histidine residues in GCS were individually replaced by other amino acids. H193A (His193 → Ala) and H193N (His193 → Asn) mutants were unaffected by 0.1 mM DEPC, a concentration that inhibited other histidine mutants and the wild-type enzyme by at least 60%. These results indicate that His193 is the primary target of DEPC and is at, or near, the UDP-Glc-binding site of GCS. His193 mutants were also insensitive to the GCS inhibitor D - threo - 1 - phenyl - 2 - decanoylamino - 3 - morpholinopropan - 1 - ol, at concentrations which inhibited the wild-type enzyme by > 80%. These results have significance for both an understanding of the GCS active site and also for the possible design of new and specific inhibitors of GCS.

scholarly journals Tackling the problem of HIV drug resistance

2016 ◽  
Vol 62 (3) ◽  
pp. 273-279
Author(s):  
Irene T. Weber ◽  
Robert W. Harrison
Keyword(s):  
Drug Resistance ◽  
Experimental Studies ◽  
Hiv Infections ◽  
Initial Step ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Practical Applications ◽  
Inhibitory Compounds ◽  
Resistant Mutants ◽  
Hiv 1

The virally-encoded HIV-1 protease is an effective target for antiviral drugs, however, treatment for HIV infections is limited by the prevalence of drug resistant viral mutants. In this review, we describe our three-pronged approach to analyze and combat drug resistance. Understanding the molecular basis for resistance due to protease inhibitors is a key initial step in this approach. This knowledge is being employed for the design of new, improved inhibitors with high affinity for resistant mutants as well as wild type enzyme. In parallel with experimental studies of diverse mutants and inhibitory compounds, we are developing efficient algorithms to predict drug resistance phenotype from genotype data. This approach has important practical applications in the clinic where genotyping is recommended for individuals with new infections.

scholarly journals Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase

2004 ◽  
Vol 379 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Hiroki KONDO ◽  
Yoriko NAKAMURA ◽  
Yi-Xin DONG ◽  
Jun-ichi NIKAWA ◽  
Shinji SUEDA
Keyword(s):  
Amino Acid ◽  
Gene Disruption ◽  
Structural Integrity ◽  
Genomic Library ◽  
Enzymic Activity ◽  
Wild Type ◽  
Tetrad Analysis ◽  
Wild Type Enzyme ◽  
Gene Coding ◽  
Responsible Gene

To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared. After transformation with a yeast genomic library, a transformant (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22t1, the RKI1 gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no. 15 was identified as the responsible gene. This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph. The site of mutation on the RKI1 gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid substitution of Arg-189 with lysine. The enzymic activity of the Arg189→Lys (R189K) mutant of ribose 5-phosphate ketolisomerase was 0.6% when compared with the wild-type enzyme. Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.

scholarly journals Heterologous expression of the allelic variant mu-class glutathione transferases μ and ψ

1991 ◽  
Vol 276 (2) ◽  
pp. 519-524 ◽  
Author(s):  
M Widersten ◽  
W R Pearson ◽  
A Engström ◽  
B Mannervik
Keyword(s):  
Amino Acid ◽  
Recombinant Proteins ◽  
Amino Acid Residue ◽  
Human Liver ◽  
Human Population ◽  
Glutathione Transferase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Allelic Variants ◽  
Isoelectric Points

cDNA encoding the more acidic form, glutathione transferase (GST) psi, of the polymorphic Mu-class GSTs discovered in liver, was mutated in the 5′-end to create an NcoI site, facilitating cloning into the expression plasmid pKK233-2. The protein expressed from this construct has a point mutation Pro-2-Ala-2, but gives a catalytically functional protein. Back-mutation of the codon for amino acid residue 2 gave rise to a plasmid expressing the wild-type enzyme GST psi, or GST Mu1b-1b. A variant cDNA, differing only in specifying lysine rather than asparagine in position 173 of the coding region, was generated by site-directed mutagenesis. The variant sequence corresponds to another cDNA clone isolated from a human liver cDNA library and expresses the near-neutral GST mu, or GST Mu1a-1a. The two recombinant proteins GST Mu1a-1a and GST Mu1b-1b, by physicochemical as well as kinetic criteria, were found to be indistinguishable from GST mu and GST psi respectively, isolated from human liver. It is therefore concluded that the recombinant proteins correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and correspond to the allelic variants observed in the human population. The two forms have different isoelectric points and their protein subunits can be separated by h.p.l.c. on a reverse-phase column. With standard substrates and inhibitors no differences in kinetic parameters between the two variants were detected. The mutated GST Mu1b-1b (Pro-2-Ala) was not significantly different in catalytic properties from the wild-type enzyme, even though Pro-2 is a well conserved amino acid residue in the known Mu-class GSTs.

scholarly journals Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides

1993 ◽  
Vol 295 (3) ◽  
pp. 645-648 ◽  
Author(s):  
M M Altamirano ◽  
J A Plumbridge ◽  
H A Barba ◽  
M L Calcagno
Keyword(s):  
Thermal Denaturation ◽  
Cysteine Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Urea Denaturation ◽  
Disulphide Bonds ◽  
Oligomeric Protein ◽  
Sds Page ◽  
Mutant Forms ◽  
Allosteric Properties

Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine residues located at positions 118, 219, 228 and 239. We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition. The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines. Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers. The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.

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