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Volumn 130, Issue 46, 2008, Pages 15467-15475

Probing general base catalysis in the hammerhead ribozyme

Author keywords

[No Author keywords available]

Indexed keywords

ALKYLATION; CATALYSIS; CHEMICAL REACTIONS; FUNCTIONAL GROUPS; HYDROCARBONS; NUCLEIC ACIDS; QUANTUM CHEMISTRY; RNA;

EID: 56449119364     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja804496z     Document Type: Article
Times cited : (34)

References (61)
  • 35
    • 56449126504 scopus 로고    scopus 로고
    • Unpublished results
    • Unpublished results.
  • 36
    • 56449124510 scopus 로고    scopus 로고
    • For a pictorial explanation of alkaline footprint interpretation for the 5′- and 3′-labeled samples, see Supporting Information.
    • For a pictorial explanation of alkaline footprint interpretation for the 5′- and 3′-labeled samples, see Supporting Information.
  • 38
    • 56449090387 scopus 로고    scopus 로고
    • Polynucleotide kinase treatment ensures the 5′-phosphorylation and 3′-dephosphorylation of all fragments. See Supporting Information for a comparison of base versus aniline catalyzed cleavage mechanisms and products.
    • Polynucleotide kinase treatment ensures the 5′-phosphorylation and 3′-dephosphorylation of all fragments. See Supporting Information for a comparison of base versus aniline catalyzed cleavage mechanisms and products.
  • 43
    • 56449107200 scopus 로고    scopus 로고
    • Initial rates were quantified in order to avoid complications due to hydrolysis of the bromoacetamide-substrate analogue at longer time points required to observe completion of the alkylation reaction
    • Initial rates were quantified in order to avoid complications due to hydrolysis of the bromoacetamide-substrate analogue at longer time points required to observe completion of the alkylation reaction.
  • 49
    • 56449121205 scopus 로고    scopus 로고
    • Manuscript in preparation
    • Manuscript in preparation.
  • 51
    • 56449086764 scopus 로고    scopus 로고
    • An important caveat must be entertained: the affinity label may perturb the hairpin ribozyme active site structure such that apparent pK a of G8 is increased relative to its pKa in the presence of native substrate. Moreover, the 2′-bromoacetamide substrate analogue is not, by definition, a mechanism based inhibitor in that its reactivity is not unmasked as a result of the catalytic mechanism. The positioning of the reactive electrophile where substrate deprotonation normally occurs encourages a mechanistic interpretation for ribozyme alkylation, especially where the alkylation rate shows log-linear increase with pH, as for hammerhead G12 alkylation. Caution must be exercised, however, as the high reactivity of the probe may lead to alkylations that reflect only fortuitous structural positioning, not the involvement of a particular residue in general base catalysis. This is exemplified by the apparently pH-independent alkylations of A6, C7, G8
    • a in the presence of native substrate. Moreover, the 2′-bromoacetamide substrate analogue is not, by definition, a mechanism based inhibitor in that its reactivity is not unmasked as a result of the catalytic mechanism. The positioning of the reactive electrophile where substrate deprotonation normally occurs encourages a mechanistic interpretation for ribozyme alkylation, especially where the alkylation rate shows log-linear increase with pH, as for hammerhead G12 alkylation. Caution must be exercised, however, as the high reactivity of the probe may lead to alkylations that reflect only fortuitous structural positioning, not the involvement of a particular residue in general base catalysis. This is exemplified by the apparently pH-independent alkylations of A6, C7, G8, and A9 in the hammerhead, which are not likely to be indicative of general base catalysis.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.