Phosphodiester bond formation

Unlike other enzymes that we have discussed, the completion of a catalytic cycle of primer extension does not result in release of the product (TP(n+1)) and recovery of the free enzyme. Instead, the product remains bound to the enzyme, in the form of a new template-primer complex, and this acts as a new substrate for continued primer extension. Catalysis continues in this way until the entire template sequence has been complemented. The overall rate of reaction is limited by the chemical steps composing cat these include the chemical step of phosphodiester bond formation and requisite conformational changes in the enzyme structure. Hence there are several potential mechanisms for inhibiting the reaction of HIV RT. Competitive inhibitors could be prepared that would block binding of either the dNTPs or the TP. Alternatively, noncompetitive compounds could be prepared that function to block the chemistry of bond formation, that block the required enzyme conformational transition(s) of turnover, or that alter the reaction pathway in a manner that alters the rate-limiting step of turnover. 

Figure 4.25 (a) DNA polymerase-catalysed phosphodiester bond formation typically requires two metal ions, usually Mg2+. (b) A model of the transition state for phosphodiester-bond formation in RNA polymerase. (From Berg et al., 2002. Reproduced with permission from W.H. Freeman and Co.)... 

The precise functions of the subunits of the core enzyme are not known. /3 is a basic (positively charged) polypeptide thought to be involved in DNA binding. The j8 subunit is the site of binding of several inhibitors of transcription and is thought to contain most or all of the active sites for phosphodiester bond formation. The a subunit is necessary for reconstituting active enzyme from separated subunits. 

Details of phosphodiester bond formation. The a, /3, and y phosphates are indicated on the initiating NTP, which in this case is ATP. The colored ovals represent NTP-binding sites on the RNA polymerase. The biochemistry of bond formation in RNA synthesis is very similar to that in DNA synthesis. 

Figure 28.2. RNA Polymerase Active Site. A model of the transition state for phosphodiester-bond formation in the active site of RNA polymerase. The 3 -hydroxyl group of the growing RNA chain attacks the a -phosphate of the incoming nucleoside triphosphate. This transition state is structurally similar to that in DNA polymerase (see Figure 27.12).

FIGURE 3.29 (a) DNA polymerase catalysed phosphodiester bond formation typically requires two metal ions, usually (b) A model of... 

DNA ligase and RNA ligase catalyze phosphodiester bond formation between 5 -phosphate and the 3 -hydroxyl ends of DNA or RNA. DNA ligases are required for DNA replication and RNA ligases are required for certain RNA splicing reactions. The function and overall reaction mechanism for DNA ligases are reviewed in Volume X of this series (80). The chemical mechanistic pathway for DNA ligase is outlined in reactions (23a)-(23c). 

Eukaryotic PNPTs are localized in the membrane of the endoplasmic reticulum (ER) where they catalyze the first step in N-linked glycoprotein biosynthesis resulting in a Dol-PP-GlcNAc intermediate. In contrast, bacterial PNPTs such as WecA, MraY, WbpL, and WbcO utilize different N-acetylhexosamine substrates and they also differ in their susceptibility to selective inhibitors. Several regions of conserved amino acid sequence can be found in bacterial and eukaryotic members of the PNPT family. It is plausible that all the members of this family utilize a common enzymatic mechanism for the formation of the phosphodiester bond. However, bacterial and eukaryotic PNPTs differ in their substrate specificity for various N-acetylhexosamine substrates and also they can discriminate the type of polyisoprenol phosphate. Und-P contains 11 isoprene units all of which are fully unsaturated, while Dol-P can be made of 15-19 isoprene units that have a saturated ct-isoprene. The ct-isoprene is the phosphorylated end of the molecule, which participates in the phosphodiester bond formation with the N-acetylhexosamine-l-P. Therefore, the ability of eukaryotic and bacterial enzymes to exquisitely discriminate their lipid substrate is likely a reflection of evolutionary divergence. 

The reaction catalyzed by RNA polymerase, (a) RNA polymerase separates the two strands of DNA and produces an RNA copy of one of the two DNA strands, (b) Phosphodiester bond formation occurs as a nucleotide is added to the growing RNA chain. 

Pulse-chase/pulse-quench experiments with KF ° indicated accumulation of the nucleotide bound enzyme species, which would not be possible if the forward reaction was much faster than the rate of conformational closing. To explain this observation, the authors proposed the presence of a kinetic road block - a slow step after the phosphodiester bond formation. However, the results of the pulse-chase/ pulse-quench experiments can also be explained by designating chemistry as the slow step, meaning that the chemical step itself plays the role of the road block. The conclusion that chemistry is a fast step in the KF reaction pathway was made based on the observation of a small thio-efifect magnitude,which, as elaborated in the following section, should not be used as a solid evidence of the chemical step being nonrate limiting. 

On the basis of negligible structural changes observed between the Pol / pre-chemistry ternary complex and the Pol [3 post-chemistry ternary complex prior to pyrophosphate release, it is important to point out that the slow fluorescence transition is not caused by the phosphodiester bond formation directly. Instead, the fluorescence change originates from a conformational change rate limited by chemistry. In other words, this conformational step should occur after chemistry with a rate faster than chemistry. 

Figure 21 Schematic representation of the free energy profiies for correct (biack) and incorrect (red) nucieotide incorporations by Poi /3. Reactions foiiow initiai binding of dNTP (1), open-to-ciosed conformationai change (2), 03 repiacing water in Mg at coordination (3a, for misincorporation oniy), proton transfer from 03 to Asp256 (3b), phosphodiester bond formation (3c), ciosed-to-open conformationai change (4), and reiease of pyrophosphate and Mg ions (5). The dashed iines represent possibie transition pathways and barriers. Adapted with permission from P. Lin V. K. Batra L. O. Pedersen W. A. Beard S. H. Wiison L. G. Pedersen, Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5670-5674. Oopyright 2008 by the Nationai Academy of Sciences.

This, in turn, accounts for the difference in the regioselectivity of phosphodiester bond formation. 

Analogues of cyclic AMP derived from the carbocyclic nucleosides aristeromycin and neplanocin have been prepared by intramolecular phosphodiester bond formation catalysed by divalent lead ions, and studies have been reported on the synthesis and biological activity of nucleoside 3, 5 -cyclic phosphotriesters and phosphoramidates. ... 

Beckman JW, Wang Q, Guengerich EP (2008) Kinetic analysis of correct nucleotide insertion by a Y-family DNA polymerase reveals conformational changes both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J Biol Chem... 

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