Nucleotidyl transfer stereochemistry

By the criteria of steady-state kinetic patterns and stereochemistry, these enzymes appear to catalyze their respective reactions by similar or closely related mechanisms. The steady-state kinetic mechanisms are of the sequential type, and in all cases so far investigated the reactions proceed with inversion of configuration at P of the nucleoside triphosphate. Thus, these reactions proceed via ternary complexes of enzyme-NTP-ROPOa , and the nucleotidyl transfer is a one-step transfer directly from the NTP to the acceptor, that is, by a single displacement at P of NTP. 

The DNA and RNA polymerase reactions, as well as the reverse transcriptase and polynucleotide phosphorylase reactions, proceed with inversion of configuration at Pa of the nucleoside triphosphate (45-50). Thus, an uneven number of displacements at phosphoms is involved in the chemical reaction mechanism, and the stereochemistry provides no evidence for the involvement of a covalent nucleotidyl-enzyme as an intermediate on the catalytic pathway. No other evidence for such an intermediate is available. Therefore, it must be concluded that the physicochemical requirements for nucleotidyl group transfer, substrate recognition, and movement along the template are derived fiom binding interactions between the enzyme and its template and substrate rather than through nucleophilic catalysis. This is also true of polynucleotide phosphorylase and other nucleotidyltransferases that catalyze reactions of polynucleotides (51, 52). 

These enzymes are not classified as nucleotidyltransferases, although they catalyze nucleotidyl group transfers in the course of activating the S -phosphoryl groups for the ligation process. The activation mechanism involves a covalent adenylyl-enzyme as an intermediate and a double displacement on of ATP (or NAD+). The chemical mechanism of the RNA ligase reaction is similar. The stereochemistry of these reactions is known for RNA ligase and is consistent with the mechanism as formulated above (81, 82). 

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