Kinetic and thermodynamic characterization of the reaction catalyzed by a polynucleotide kinase ribozyme.
We have previously isolated a series of ribozymes with polynucleotide kinase activity [Lorsch, J.R., & Szostak, J.W. (1994) Nature 371, 31-36]. In order to learn how such newly evolved RNAs effect catalysis, we have determined a number of the kinetic and thermodynamic parameters for the reaction catalyzed by one of these ribozymes. This ribozyme, a class I polynucleotide kinase, catalyzes the transfer of the gamma-(thio)phosphate from ATP(-gamma S) to the 5'-hydroxyl of a 7-mer oligoribonucleotide. The kcat for the reaction with ATP-gamma S is 0.17 min-1 with a Km of approximately 3 mM. The Km for the oligoribonucleotide substrate 5'-HO-GGAACCU-3' is 2 microM, the same as the Kd for this substrate in the presence or absence of ATP-gamma S. Neither the binding of substrates nor the release of products is the rate-limiting step of the reaction. The binding of substrates and release of products appear to occur in a random fashion, with no synergy of binding between the ATP(-gamma S) and oligoribonucleotide substrates. The ribozyme binds the oligoribonucleotide substrate no more strongly than would be expected for the formation of a simple RNA-RNA duplex, suggesting that there are no tertiary contacts between the ribozyme and the RNA substrate. The oligoribonucleotide substrate binding site has been located, and the sequence specificity of the ribozyme could be altered by mutating this binding site. The ribozyme is specific for adenosine triphosphate substrates; GTP-gamma S reacts approximately 650-fold slower than ATP-gamma S. With ATP as the substrate, the Kms remain unchanged, but kcat decreases by a factor of 50, consistent with a rate-limiting chemical step occurring through a dissociative transition state. The pH independence (from pH 5.5 to 8.5) of kcat/Km and of the rate constant for the conversion of the ternary substrate complex into the ternary products complex is also consistent with a dissociative phosphoryl transfer mechanism. These results suggest that this newly evolved catalyst operates in a relatively simple manner, with independent substrate binding sites and without changing the mechanism of the underlying chemical reaction.
Lorsch
J R
JR
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
Szostak
J W
JW
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D009843:Oligoribonucleotides; D016337:RNA, Catalytic; C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase