Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme.
The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu(2+) for optimal catalysis of thiophosphoryl transfer from GTPγS. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu(2+), indicating a specific catalytic role independent of any potential interactions with the GTPγS thiophosphoryl group. In-line probing and ATPγS competition both argue against direct Cu(2+) binding by RNA; rather, these data establish that Cu(2+) enters the active site within a Cu(2+)•GTPγS or Cu(2+)•GTP chelation complex, and that Cu(2+)•nucleobase interactions further enforce Cu(2+) selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg(2+) with [Co(NH3)6](3+) significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg(2+) coordination is structural rather than catalytic. Replacing Mg(2+) with alkaline earths of increasing ionic radii (Ca(2+), Sr(2+) and Ba(2+)) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step.
Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri School of Medicine, Columbia, MO 65211, USA.
Elisa
E
Biondi
Raghav R
RR
Poudyal
Joshua C
JC
Forgy
Andrew W
AW
Sawyer
Adam W R
AW
Maxwell
Donald H
DH
Burke
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D001483:Base Sequence; D002021:Buffers; D002384:Catalysis; D020134:Catalytic Domain; D056831:Coordination Complexes / Q000737:chemistry*; D003300:Copper / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D058116:Lewis Acids; D008274:Magnesium / Q000737:chemistry; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*
D002021:Buffers; D056831:Coordination Complexes; D058116:Lewis Acids; D016337:RNA, Catalytic; C022571:adenosine 5'-O-(3-thiotriphosphate); D003300:Copper; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D008274:Magnesium