Polymerase catalysis

Substrate selection by polymerases determines, to a large extent, the accuracy of genomic replication. The efficiency of nucleotidyl transfer is dependent upon several interrelated factors. The two-metal-ion-dependent mechanism of polymerase catalysis appears to be universally conserved in most respects, with... 

Figure 13.8 Proposed reaction mechanism for DNA polymerase catalysis. The polymerase active site contains three carhoxylate residues and probably a lysine. The three carboxylate side chains anchor a pair of divalent metal ions (e.g. Mg ). In the proposed mechanism, two carboxy-lates coordinate directly to the two Mg, one of which promotes the deprotonation of the 3 -OH of the primer. An in-hne attack of the a-phosphorus atom of dNTP forms a bipyramidal pentaco-ordinated oxyphosphorane transition state with the in-coming and departing atoms at apical positions. The possible involvement of a third Mg coordinated to P- and y-phosphates is also shown...

Perez-Baena et al. reported SCNPs that contained a tris(penta-fluorophenyl)boron (B(C6F6)3) active site to mimic the function of reductase and polymerase. Catalysis with this SCNPs could only be carried out in halogenated solvents, toluene, and benzene. Solvents that could form adducts with boron were unsuitable, as they quenched the catalyst. The kinetics and TOP of catalysis by these particles were influenced by size, composition, quantity, and location of the active sites in the SCNPs. The TOP was also proportional to the molecular weight and inversely proportional to the hydrodynamic radius of the SCNPs. 

RTases have four conserved motifs centering on or around such amino acids as Asp, Gly, (Tyr-X-)-Asp-Asp, and Lys (83,84). The palm subdomain of p66 contains the catalytic triad (Asp-110, Asp-185, and Asp-186) which structurally superimposes on the catalytically important Asp-705, Asp-882, and Glu-883 of the Klenow polymerase and also on the three essential Asp residues [150, 224, and 225] of the MoLV RTase. The Asp-224 and -225 of the MoLV RTase are coordinated to a metal ion (Mn ) which is presumed to play a key role in polymerase catalysis (85). Site-specific mutations of each Asp residue in the Asp -Asp motif to Asn or Gin render the RTase catalytically inactive, although the mutant enzymes can still bind the template-primer complex (86). The mutation of Asp-110 also abolishes the RTase activity. The ddNTP-resistant mutations... 

Poly(L-malate) decomposes spontaneously to L-ma-late by ester hydrolysis [2,4,5]. Hydrolytic degradation of the polymer sodium salt at pH 7.0 and 37°C results in a random cleavage of the polymer, the molecular mass decreasing by 50% after a period of 10 h [2]. The rate of hydrolysis is accelerated in acidic and alkaline solutions. This was first noted by changes in the activity of the polymer to inhibit DNA polymerase a of P. polycephalum [4]. The explanation of this phenomenon was that the degradation was slowest between pH 5-9 (Fig. 2) as would be expected if it were acid/base-catalyzed. In choosing a buffer, one should be aware of specific buffer catalysis. We found that the polymer was more stable in phosphate buffer than in Tris/HCl-buffer. 

Substances that do not target the active site but display inhibition by allosteric mechanisms are associated with a lower risk of unwanted interference with related cellular enzymes. Allosteric inhibition of the viral polymerase is employed in the case of HIV-1 nonnucleosidic RT inhibitors (NNRTl, see chapter by Zimmermann et al., this volume) bind outside the RT active site and act by blocking a conformational change of the enzyme essential for catalysis. A potential disadvantage of targeting regions distant from the active site is that these may be subject to a lower selective pressure for sequence conservation than the active site itself, which can lower the threshold for escape of the virus by mutation. 

There are three mechanistic possibilities for catalysis by two-metal ion sites (Fig. 10). The first of these is the classic two-metal ion catalysis in which one metal plays the dominant role in activating the substrate toward nucleophilic attack, while the other metal ion furnishes the bound hydroxide as the nucleophile (Fig. 10 a). Upon substrate binding, the previously bridged hydroxide shifts to coordinate predominately with one metal ion. Enzymes believed to function through such a mechanism include a purple acid phosphatase [79], DNA polymerase I [80], inositol monophosphatase [81],fructose-1,6-bisphosphatase [82], Bam HI [83], and ribozymes [63]. 

Kun E, Kirsten E, Ordahl CP (2002) Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone HI trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I). J Biol Chem 277 39066-39069 Kun E, Kirsten E, Mendeleyev J, Ordahl CP (2004) Regulation of the enzymatic catalysis of poly(ADP-ribose) polymerase by dsDNA, polyamines, Mg2-F, Ca2-F, histones HI and H3, and ATP. Biochemistry 43 210-216... 

Currently, only a handful of examples of unique protein carboxylate-zinc interactions are available in the Brookhaven Protein Data Bank. Each of these entries, however, displays syn coordination stereochemistry, and two are bidentate (Christianson and Alexander, 1989) (Fig. 5). Other protein structures have been reported with iyw-oriented car-boxylate-zinc interactions, but full coordinate sets are not yet available [e.g., DNA polymerase (Ollis etal., 1985) and alkaline phosphatase (Kim and Wyckoff, 1989)]. A survey of all protein-metal ion interactions reveals that jyw-carboxylate—metal ion stereochemistry is preferred (Chakrabarti, 1990a). It is been suggested that potent zinc enzyme inhibition arises from syn-oriented interactions between inhibitor carboxylates and active-site zinc ions (Christianson and Lipscomb, 1988a see also Monzingo and Matthews, 1984), and the structures of such interactions may sample the reaction coordinate for enzymatic catalysis in certain systems (Christianson and Lipscomb, 1987). 

Several other major classes of enzymes, among them the nucleic acid polymerases, activate ATP (and other NTPs) in a completely different manner. Similar to transphos-phoiylation enzymes, they utilize two metal ions for catalysis. However, steric interactions are purposely employed in order to reverse the preferred binding situation. A MaMp y motif is generated which weakens the P —O—P,5 linkage This allows a nucleoside monophosphate group to be transferred (under liberation of PPi), a process which is essential in the biosynthesis of DNA and RNA sequences. 

Some of the polymerases exist as single polypeptide chains, while others function only as large complexes. In every case a two-metal ion catalytic mechanism with in-line nucleotidyl transfer,269 illustrated in Fig. 27-13, appears to be used by the enzymes.267 270 Two-metal ion catalysis is also observed for phosphatases and ribozymes (Chapter 12). 

Figure 27-13 Proposed mechanism and transition state structure for the synthetic nucleotidyltransfer activity of DNA polymerase 3 (and other DNA polymerases). The chain-terminating inhibitor dideoxy CTP is reacting with the 3 -OH group of a growing polynucleotide primer chain. This -OH group (as -0 ) makes an in-line nucleophilic attack on Pa of the dideoxy-CTP. Notice the two metal ions, which interact with the phospho groups and which are held by three aspartate side chains. Two of the latter, Asp 190 and Asp 256, are present in similar positions in all of the polymerases. The active centers for the hydrolytic 3 -5 and 5 -3 exonuclease activities of some of the polymerases also appear to involve two-metal catalysis and in-line displacement. See Sawaya et al.27i...

Mullis, K.B. (1990). The unusual origin of the polymerase chain reaction. Sci. Am. 262(4), 36-43. Narlikar, G.J. Herschlag, D. (1997). Mechanistic aspects of enzymatic catalysis Lessons from comparisons of RNA and protein enzymes. Annu. Rev. Biochem. 66,19-59. 

DNA polymerases catalyze DNA synthesis in a template-directed manner (Box 16). For most known DNA polymerases a short DNA strand hybridized to the template strand is required to serve as a primer for initiation of DNA synthesis. Nascent DNA synthesis is promoted by DNA polymerases by catalysis of nucleophilic attack of the 3 -hydroxyl group of the 3 -terminal nucleotide of the primer strand on the a-phosphate of an incoming nucleoside triphosphate (dNTP), leading to substitution of pyrophosphate. This phosphoryl transfer step is promoted by two magnesium ions that stabilize a pentacoordinated transition state by complex-ation of the phosphate groups and essential carboxylate moieties in the active site (Figure 4.1.1) [2],... 

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