E:p/t:dNTP complex

Direct observation of the E p/t dNTP complex was obtained using pulse-chase experiments. In such experiments, incorporation of labeled nucleotide to an E p/t complex is either quenched by the addition of HC1 or allowed to proceed after the addition of a large excess of cold unlabeled dNTP (the chase step) followed by acid quench. In the HC1 quench experiments, the acid quenches all the enzyme-bound species. On the other hand, when the reaction is chased with cold dNTP, each of the enzyme-bound species is allowed to partition both in the forward and reverse directions. The amount of partitioning in the forward direction is observed as an excess of labeled product, compared with the acid quench experiment, while the dNTP that partitions in the reverse direction is diluted and remains unobservable. As an excess was observed and because the binding of dNTP to the E p/t complex is rapid, the observed flux or excess is mainly due to the E p/t dNTP complex. 

Nucleotide incorporation into the enzyme-p/t complex is initiated by the binding of a dNTP to the E p/t complex to form the E p/t dNTP complex (Step 2 Fig. 1). 

VII. Conformational Transition to a Catalytically Active Ternary Complex The E p/t dNTP Complex... 

The rate-limiting step in the kinetic pathway of nucleotide incorporation is the conversion of the E p/t dNTP complex to the activated complex, E p/t dNTP (Step 3 in Fig. 1). This step is crucial in many respects. First, it is essential for the phosphoryl transfer reaction to occur. During the E p/t dNTP to E p/t dNTP transition, all the components of the active site are assembled and organized in a topological and geometrical arrangement that allows the enzyme to proceed with the chemical step (Step 4). Second, Step 3 plays a major role in the mechanism of discrimination between correct versus incorrect nucleotides. Interpretation of the kinetic measurements has led to the hypothesis that the E p/t dNTP... 

Recent kinetic work on RB69 polymerase (family B) and structural comparison between the RB69 polymerase and other polymerase families led to the postulation of a different initial binding event for dNTP (Yang et al, 2002a). The crystal structure of the closed ternary E p/t ddNTP complex of the RB69 polymerase (see Section VII) shows interactions between the fingers subdomain and the ddNTP s triphosphate moiety... 

It has been shown for the Klenow fragment that the PPi product has only a fivefold lower affinity for the E p/t complex than dNTPs, suggesting that the product of the reaction could compete for binding of dNTPs (Kuchta et al., 1987). However, for T7 DNA polymerase, the affinity of PPi is extremely low and is nowhere near comparable to affinities for correct nucleotide binding, differing by nearly a factor of 1000 (Patel et al, 1991). T4 has similarly reduced affinities for PPi compared with Klenow, being in the low millimolar range (Capson et al, 1992). 

---