GSBS Student Publications

Title

Ribozyme-catalyzed dipeptide synthesis in monovalent metal ions alone

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Molecular Genetics and Microbiology; Program in Molecular Medicine

Date

3-3-2007

Document Type

Article

Medical Subject Headings

Catalysis; Cations, Divalent; Cations, Monovalent; Dipeptides; Hydrogen-Ion Concentration; Metals; Peptide Biosynthesis, Nucleic Acid-Independent; RNA, Catalytic

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

Previously we have identified a highly active ribozyme (R180, cis ribozyme) that can catalyze dipeptide synthesis using N-biotinylcaproyl-aminoacyl-adenylate anhydride (Bio-aa-5'-AMP) as its substrate. In this work, we re-engineered the cis R180 ribozyme into a 158-nt trans ribozyme (TR158) and designed a new substrate (5'-Phe-linker-20-mer). First, the metal ion requirements were examined and compared between the two ribozymes. Both R180 and TR158 ribozymes were active in Mg2+ and Ca2+ but inert with Zn2+, Cu2+, Mn2+, and Co2+. It is intriguing that both ribozymes were highly active in Li+, Na+, or K+ alone but showed very low activity with NH4+. The two ribozymes showed similar linear concentration dependence on Li+ and K+, while they displayed different dependency behavior on Mg2+. Moreover, by using the trans system, the detailed kinetic studies and pH dependent experiments were performed in either 10 mM Mg2+ or 1.0 M Li+. Analysis of kcat and Km values obtained at different pHs (6.0 to 9.0) indicated that it is the catalytic activity of the ribozyme but not the substrate binding affinity that changes significantly with pH. The slopes of the linear parts of the pH-rate plots were close to 1.0 in both Mg2+- and Li+-mediated reactions, suggesting that one proton transfer is involved in the rate-limiting step of catalysis. Overall, our results suggest that Mg2+ and Li+ function similarly in the ribozyme-catalyzed dipeptide synthesis.

Rights and Permissions

Citation: Biochemistry. 2007 Mar 27;46(12):3714-23. Epub 2007 Mar 2. Link to article on publisher's site

Related Resources

Link to Article in PubMed

Journal Title

Biochemistry

PubMed ID

17330961