N Acetylimidazole

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Terence L. Kirley - One of the best experts on this subject based on the ideXlab platform.

  • IdeNtificatioN of a tyrosiNe residue respoNsible for N-Acetylimidazole-iNduced iNcrease of activity of ecto-Nucleoside triphosphate diphosphohydrolase 3. PuriNergic SigNalliNg 2005
    2013
    Co-Authors: Saswata Basu, Terence L. Kirley
    Abstract:

    site-directed mutageNesis, tyrosiNe modificatioN Chemical modificatioN iN combiNatioN with site-directed mutageNesis was used to ideNtify a tyrosiNe residue respoNsible for the iNcrease iN ecto-Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) Nucleotidase activity after acetylatioN with a tyrosiNe-selective reageNt, N-Acetylimidazole. The NTPDase3 ATPase activity is iNcreased more thaN the ADPase activity by this reageNt. Several fairly well coNserved tyrosiNe residues (252, 255, aNd 262) that are located iN or very Near apyrase coNserved regioN 4a (ACR4a) were mutated. These mutaNts were all active, but mutatioN of tyrosiNe 252 to either alaNiNe or pheNylalaNiNe elimiNated the activity iNcrease observed after N-Acetylimidazole treatmeNt of the wild-type eNzyme. This suggests that the acetylatioN of tyrosiNe 252 is respoNsible for the iNcreased activity. StabilizatioN of quaterNary structure has resulted iN iNcreased eNzyme activities for the NTPDases. However, mutatioN of these three tyrosiNe residues did Not result iN global chaNges of tertiary or quaterNary structure, as measured by CibacroN blue biNdiNg, chemical cross liNkiNg, aNd Native gel electrophoretic aNalysis. Nevertheless, disruptioN of the oligomeric structure with the detergeNt TritoN X-100 abolished the iNcrease iN activity iNduced by this reageNt. IN additioN, mutatioNs that abolished the N-Acetylimidazole effect also atteNuated the iNcreases of eNzyme activity observed after lectiN aNd chemical cross-liNkiNg treatmeNts, which were previously attributed to stabilizatioN of the quaterNary structure. Thus, we speculate that the acetylatioN of tyrosiNe 252 might iNduce a subtle coNformatioNal chaNge iN NTPDase3, resultiNg iN the observed iNcrease iN activity

  • IdeNtificatioN of a tyrosiNe residue respoNsible for N-Acetylimidazole-iNduced iNcrease of activity of ecto-Nucleoside triphosphate diphosphohydrolase 3
    Purinergic Signalling, 2005
    Co-Authors: Saswata Basu, Terence L. Kirley
    Abstract:

    Chemical modificatioN iN combiNatioN with site-directed mutageNesis was used to ideNtify a tyrosiNe residue respoNsible for the iNcrease iN ecto-Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) Nucleotidase activity after acetylatioN with a tyrosiNe-selective reageNt, N -Acetylimidazole. The NTPDase3 ATPase activity is iNcreased more thaN the ADPase activity by this reageNt. Several fairly well coNserved tyrosiNe residues (252, 255, aNd 262) that are located iN or very Near apyrase coNserved regioN 4a (ACR4a) were mutated. These mutaNts were all active, but mutatioN of tyrosiNe 252 to either alaNiNe or pheNylalaNiNe elimiNated the activity iNcrease observed after N -Acetylimidazole treatmeNt of the wild-type eNzyme. This suggests that the acetylatioN of tyrosiNe 252 is respoNsible for the iNcreased activity. StabilizatioN of quaterNary structure has resulted iN iNcreased eNzyme activities for the NTPDases. However, mutatioN of these three tyrosiNe residues did Not result iN global chaNges of tertiary or quaterNary structure, as measured by CibacroN blue biNdiNg, chemical cross liNkiNg, aNd Native gel electrophoretic aNalysis. Nevertheless, disruptioN of the oligomeric structure with the detergeNt TritoN X-100 abolished the iNcrease iN activity iNduced by this reageNt. IN additioN, mutatioNs that abolished the N -Acetylimidazole effect also atteNuated the iNcreases of eNzyme activity observed after lectiN aNd chemical cross-liNkiNg treatmeNts, which were previously attributed to stabilizatioN of the quaterNary structure. Thus, we speculate that the acetylatioN of tyrosiNe 252 might iNduce a subtle coNformatioNal chaNge iN NTPDase3, resultiNg iN the observed iNcrease iN activity.

Julie L. Waddle - One of the best experts on this subject based on the ideXlab platform.

Saswata Basu - One of the best experts on this subject based on the ideXlab platform.

  • IdeNtificatioN of a tyrosiNe residue respoNsible for N-Acetylimidazole-iNduced iNcrease of activity of ecto-Nucleoside triphosphate diphosphohydrolase 3. PuriNergic SigNalliNg 2005
    2013
    Co-Authors: Saswata Basu, Terence L. Kirley
    Abstract:

    site-directed mutageNesis, tyrosiNe modificatioN Chemical modificatioN iN combiNatioN with site-directed mutageNesis was used to ideNtify a tyrosiNe residue respoNsible for the iNcrease iN ecto-Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) Nucleotidase activity after acetylatioN with a tyrosiNe-selective reageNt, N-Acetylimidazole. The NTPDase3 ATPase activity is iNcreased more thaN the ADPase activity by this reageNt. Several fairly well coNserved tyrosiNe residues (252, 255, aNd 262) that are located iN or very Near apyrase coNserved regioN 4a (ACR4a) were mutated. These mutaNts were all active, but mutatioN of tyrosiNe 252 to either alaNiNe or pheNylalaNiNe elimiNated the activity iNcrease observed after N-Acetylimidazole treatmeNt of the wild-type eNzyme. This suggests that the acetylatioN of tyrosiNe 252 is respoNsible for the iNcreased activity. StabilizatioN of quaterNary structure has resulted iN iNcreased eNzyme activities for the NTPDases. However, mutatioN of these three tyrosiNe residues did Not result iN global chaNges of tertiary or quaterNary structure, as measured by CibacroN blue biNdiNg, chemical cross liNkiNg, aNd Native gel electrophoretic aNalysis. Nevertheless, disruptioN of the oligomeric structure with the detergeNt TritoN X-100 abolished the iNcrease iN activity iNduced by this reageNt. IN additioN, mutatioNs that abolished the N-Acetylimidazole effect also atteNuated the iNcreases of eNzyme activity observed after lectiN aNd chemical cross-liNkiNg treatmeNts, which were previously attributed to stabilizatioN of the quaterNary structure. Thus, we speculate that the acetylatioN of tyrosiNe 252 might iNduce a subtle coNformatioNal chaNge iN NTPDase3, resultiNg iN the observed iNcrease iN activity

  • IdeNtificatioN of a tyrosiNe residue respoNsible for N-Acetylimidazole-iNduced iNcrease of activity of ecto-Nucleoside triphosphate diphosphohydrolase 3
    Purinergic Signalling, 2005
    Co-Authors: Saswata Basu, Terence L. Kirley
    Abstract:

    Chemical modificatioN iN combiNatioN with site-directed mutageNesis was used to ideNtify a tyrosiNe residue respoNsible for the iNcrease iN ecto-Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) Nucleotidase activity after acetylatioN with a tyrosiNe-selective reageNt, N -Acetylimidazole. The NTPDase3 ATPase activity is iNcreased more thaN the ADPase activity by this reageNt. Several fairly well coNserved tyrosiNe residues (252, 255, aNd 262) that are located iN or very Near apyrase coNserved regioN 4a (ACR4a) were mutated. These mutaNts were all active, but mutatioN of tyrosiNe 252 to either alaNiNe or pheNylalaNiNe elimiNated the activity iNcrease observed after N -Acetylimidazole treatmeNt of the wild-type eNzyme. This suggests that the acetylatioN of tyrosiNe 252 is respoNsible for the iNcreased activity. StabilizatioN of quaterNary structure has resulted iN iNcreased eNzyme activities for the NTPDases. However, mutatioN of these three tyrosiNe residues did Not result iN global chaNges of tertiary or quaterNary structure, as measured by CibacroN blue biNdiNg, chemical cross liNkiNg, aNd Native gel electrophoretic aNalysis. Nevertheless, disruptioN of the oligomeric structure with the detergeNt TritoN X-100 abolished the iNcrease iN activity iNduced by this reageNt. IN additioN, mutatioNs that abolished the N -Acetylimidazole effect also atteNuated the iNcreases of eNzyme activity observed after lectiN aNd chemical cross-liNkiNg treatmeNts, which were previously attributed to stabilizatioN of the quaterNary structure. Thus, we speculate that the acetylatioN of tyrosiNe 252 might iNduce a subtle coNformatioNal chaNge iN NTPDase3, resultiNg iN the observed iNcrease iN activity.

Jesper Z. Haeggström - One of the best experts on this subject based on the ideXlab platform.

  • Chemical modificatioN of leukotrieNe A4 hydrolase. INdicatioNs for esseNtial tyrosyl aNd argiNyl residues at the active site.
    Biochemistry, 1995
    Co-Authors: Martin J Mueller, Bengt Samuelsson, Jesper Z. Haeggström
    Abstract:

    : We have employed chemical modificatioN to ideNtify amiNo acids esseNtial for the catalytic activities of the bifuNctioNal ziNc metalloeNzyme leukotrieNe A4 hydrolase (EC 3.3.2.6). The epoxide hydrolase aNd the peptidase activity were both rapidly iNactivated by N-Acetylimidazole aNd tetraNitromethaNe. Furthermore, treatmeNt with 2,3-butaNedioNe aNd pheNylglyoxal also resulted iN loss of both activities. LeukotrieNe A4 hydrolase could be protected from iNactivatioN by these tyrosyl aNd argiNyl reageNts by the competitive iNhibitors bestatiN aNd captopril, respectively. Two tyrosyl aNd three argiNyl residues were fouNd by differeNtial labeliNg techNiques to be protected by the iNhibitors, which thus suggested that these amiNo acids are located close to or at the active ceNter of the eNzyme. Limited modificatioN by thiol reageNts aNd particularly methyl methaNethiosulfoNate led to a > 10-fold iNcrease iN the peptidase activity aNd a decreased epoxide hydrolase activity, whereas proloNged treatmeNt iNhibited both activities. KiNetic aNalysis of modified eNzyme, usiNg the substrate alaNiNe p-NitroaNilide, revealed that the stimulatory effect oN the peptidase activity was due to iNcreased eNzyme displayed a reduced appareNt affiNity coNstaNt for chloride ioNs, which stroNgly stimulate the peptidase activity. Neither activatioN Nor iNactivatioN by methyl methaNethiosulfoNate was iNflueNced by the preseNce of competitive iNhibitors, which suggested that this compouNd did Not react with amiNo acids at the active ceNter but rather with residues of importaNce for the overall eNzyme coNformatioN.

Lawrence J Marnett - One of the best experts on this subject based on the ideXlab platform.

  • acetylatioN of prostaglaNdiN eNdoperoxide syNthase by N Acetylimidazole comparisoN to acetylatioN by aspiriN
    Biochemistry, 1992
    Co-Authors: Isabelle Wells, Lawrence J Marnett
    Abstract:

    TreatmeNt of prostaglaNdiN eNdoperoxide (PGH) syNthase apoproteiN with a 100- or 1000-fold excess of N-Acetylimidazole (NAI) led to time-depeNdeNt iNactivatioN of both cyclooxygeNase aNd peroxide activities. RecoNstitutioN of apoproteiN with heme prior to iNcubatioN with NAI substaNtially protected the eNzyme from iNactivatioN. PretreatmeNt of the proteiN with either acetylsalicylic acid (aspiriN) or (+/-)-2-fluoro-alpha-methyl-4-bipheNylacetic acid (flurbiprofeN), which iNhibit cyclooxygeNase activity, did Not alter the time course of peroxidase iNactivatioN by NAI. TreatmeNt of NAI-iNactivated apoPGH syNthase with hydroxylamiNe led to substaNtial regeNeratioN of both cyclooxygeNase aNd peroxidase activities. QuaNtitatioN of radioactivity followiNg iNcubatioN of PGH syNthase with [3H-acetyl]NAI iNdicated iNcorporatioN of 1.7 +/- 0.9 acetyl groups/70-kDa subuNit. Cleavage of acetylated proteiN with trypsiN uNder NoNdeNaturiNg coNditioNs followed by high-performaNce liquid chromatography aNalysis demoNstrated that most of the radioactivity was iNcorporated iNto the 33-kDa fragmeNt although sigNificaNt radioactivity was also detectable iN the 38-kDa fragmeNt. Chymotryptic peptide mappiNg of acetylated proteiN revealed Numerous poteNtial sites of acetylatioN distributed iN widely divergeNt regioNs of the proteiN. No appareNt differeNces were observed betweeN the chymotryptic maps of apo- aNd holoeNzyme, suggestiNg that the adduct respoNsible for loss of catalytic activity is uNstable to the chromatographic coNditioNs. The differeNt biochemical properties of PGH syNthase acetylated by NAI or aspiriN suggest that a major determiNaNt of the specificity of aspiriN for Ser530 is biNdiNg of the salicylate moiety to this regioN of the PGH syNthase proteiN.