Allosteric regulation of a ribozyme activity through ligand-induced conformational change
Open Access
- 1 July 1998
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 26 (14), 3379-3384
- https://doi.org/10.1093/nar/26.14.3379
Abstract
An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and a flavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5–6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent kcat of Rz3 increases by ∼10-fold upon addition of saturating amounts of FMN. To determine the rate constants(Km4 and kcat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the kcat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.Keywords
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