Effects of cetyltrimethylammonium bromide on reactions catalyzed by maxizymes, a novel class of metalloenzymes.
We demonstrated previously that some shortened forms of hammerhead ribozymes had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme. Moreover, the active species appeared to form dimeric structures with a common stem II (in order to distinguish monomeric forms of conventional minizymes that have low activity from our novel dimers with high-level activity, the latter very active short ribozymes were designated 'maxizymes'). The dimers can be homodimeric (with two identical binding sequences) or heterodimeric (with two different binding sequences). In the case of heterodimers, they are in equilibrium with inactive homodimers. In this study, we investigated the effects of cationic detergent, cetyltrimethylammonium bromide (CTAB), on reactions catalyzed by a variety of maxizymes. The slope of close to unity in profiles of pH versus rate demonstrated that the deprotonation was important in catalysis and that the rate-limiting chemical step was followed in these reactions. Addition of appropriate amounts of CTAB enhanced the activity of a variety of maxizymes. The activity of our least stable, least active maxizyme was enhanced 100-fold by CTAB. Thus, CTAB effectively enhanced the conversion of kinetically trapped inactive conformations to active forms. Moreover, we suggest that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of appropriate amounts of CTAB.
Nakayama
A
A
National Institute for Advanced Interdisciplinary Research, Tsukuba Science City, Japan.
Warashina
M
M
Kuwabara
T
T
Taira
K
K
D020029:Base Pairing / Q000187:drug effects; D001665:Binding Sites; D002384:Catalysis / Q000187:drug effects; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D015497:HIV-1 / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000187:drug effects* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
D002593:Cetrimonium Compounds; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger; D008274:Magnesium; D000077286:Cetrimonium