Nucleotide substrate

Crystal structures of the NS5B polymerase alone and in complexes with nucleotide substrates have been solved and applied to discovery programs (Ago et al. 1999 Bressanelli et al. 2002 Bressanelli et al. 1999 Lesburg et al. 1999 O Farrell et al. 2003). From these studies, HCV polymerase reveals a three-dimensional structure that resembles aright hand with characteristic fingers, palm, and thumb domain, similar to the architectures of the RNA polymerases of other viruses. However, none of these experimental structures contained the ternary initiation complex with nu-cleotide/primer/template, as obtained with HIV RT. Accordingly, HCV initiation models have been built using data from other viral systems in efforts to explain SAR (Kozlov et al. 2006 Yan et al. 2007). 

Ribonucleotide reductase is required for the formation of the deoxyribonucleotides for DNA synthesis. Figure 1-18-2 shows its role in dTMP synthesis, and Figure 1-18-3 shows all four nucleotide substrates ... 

The equilibrium constant of an enzyme-catalyzed reaction can depend greatly on reaction conditions. Because most substrates, products, and effectors are ionic species, the concentration and activity of each species is usually pH-dependent. This is particularly true for nucleotide-dependent enzymes which utilize substrates having pi a values near the pH value of the reaction. For example, both ATP" and HATP may be the nucleotide substrate for a phosphotransferase, albeit with different values. Thus, the equilibrium constant with ATP may be significantly different than that of HATP . In addition, most phosphotransferases do not utilize free nucleotides as the substrate but use the metal ion complexes. Both ATP" and HATP have different stability constants for Mg +. If the buffer (or any other constituent of the reaction mixture) also binds the metal ion, the buffer (or that other constituent) can also alter the observed equilibrium constant . ... 

This enzyme [EC 2.7.7.44] catalyzes the reaction of UTP with l-phospho-a-D-glucuronate to produce llDP-o-glu-curonate and pyrophosphate. CTP can also serve as the nucleotide substrate, albeit not as effectively. 

This enzyme [EC 2.7.1.36] catalyzes the reaction of ATP with (i )-mevalonate to produce ADP and (i )-5-phos-phomevalonate. The nucleotide substrate can also be provided with CTP, GTP, or UTP. 

This enzyme [EC 2.7.1.77] catalyzes the reaction of a nucleotide with a 2 -deoxynucleoside to produce a nucleoside and a 2 -deoxynucleoside 5 -monophosphate. The nucleotide substrate can be substituted with phenyl phosphate and nucleoside 3 -phosphates, although they are not as effective. 

This enzyme [EC 2.7.1.11], also known as phosphohexo-kinase and phosphofructokinase 1, catalyzes the reaction of ATP with D-fructose 6-phosphate to produce ADP and D-fructose 1,6-bisphosphate. Both D-tagatose 6-phosphate and sedoheptulose 7-phosphate can act as the sugar substrate. UTP, CTP, GTP, and ITP all can act as the nucleotide substrate. This enzyme is distinct from that of 6-phosphofructo-2-kinase. See also ATP GTP Depletion... 

Positional isotope exchange ( PIX ) is a very valuable technique in determining enzyme mechanisms, particularly those utilizing ATP, GTP, or another NTP substrate -. For example, the nucleotide substrate can be labeled with in bridging (i.e., P—O—P or phos-phoanhydride oxygen) and/or its nonbridging positions. 

Succinyl-CoA synthetase (GDP) [EC 6.2.1.4], also known as succinate CoA ligase, catalyzes the reversible reaction of GTP with succinate and coenzyme A to produce GDP, succinyl-CoA, and orthophosphate. The nucleotide substrate can be replaced with ITP and itaconate can substitute for succinate. 

Williams, R.L. Oren, D.A. Munoz-Dorado, J. Inoue, S. Inoue, M. Arnold, E. Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution. J. Mol. Biol., 234, 1230-1247 (1993)... 

The structures of the native enzyme and its complexes with several inhibitors have since been obtained at higher resolution in other laboratories, to afford a more complete description of the enzyme - substrate interactions.191 Particularly noteworthy are the lysine residues 7,41, and 66. That these are an important part of the catalytic machinery has been deduced from their conservation in evolution (they have been found in all homologous ribonucleases that have been sequenced), and from their loss of activity when they are acetylated. Lys-41 is particularly important. The lysine side chains are very mobile in the free enzyme, but their mobilities are much decreased on the binding of nucleotide substrate analogues. Lys-41 interacts directly with the phosphate moiety and is thought to stabilize the pentacovalent intermediate. Another residue that has been con-... 

Kinetic Constants for the Nucleotide Substrates of Yeast Hexokinase... 

Flaromy TP, Raleigh J, Sundaralingam M (1980) Enzyme-bound conformations of nucleotide substrates. X-ray structure and absolute configuration of 8,5 -cycloadenosine monohydrate. Biochemistry 19 1718-1722... 

Shah, A. M., Maitra, M., and Sweasy, J. B. (2003). Variants of DNA polymerase P extend mispaired DNA due to increased affinity for nucleotide substrate. Biochemistry 42, 10709-10717. 

We focus on position 75 to limit PyC incorporation to one well-defined location. In certain cases, it may be beneficial to incorporate PyC to both positions C74 and C75 in order to enhance the fluorescence emission signal of the probe. This is achieved by using a tRNA primer terminated at position 73 and by extending the primer with PyCTP and ATP as the nucleotide substrates. In the absence of normal CTP, complete extension of the primer from position 74 to 76, as visualized by denaturing gel analysis (e.g., Fig. 4.3C), is an indication that PyC has been incorporated at both positions. However, due to the rapid reaction of the CCA enzyme to synthesize consecutive C74 and C75 (Dupasquier et al., 2008), it would be difficult to incorporate PyC to just position 74. 

A high-throughput assay for bacterial RNA polymerase has been successfully developed and validated using a 96-well, automated format [70], The reaction mixture contained a DNA template, nucleotide substrates (NTPs), supplemented with a-33P-labeled CTP in Tris-acetate buffer (pH 6.8). The polymerase reaction was carried out at 34°C for 40 min (providing linear kinetics). The effect of dimethylsulfoxide (DMSO), the usual solvent for test compounds used in a screen, was taken into consideration. The radiolabeled RNA transcripts were allowed to bind diethyl aminoethyl (DEAE) beads, which were then separated via filtration, and radioactivity associated with the wells was quantitated to measure the RNA polymerase activity. The standard deviation of the measured activity was typically < 15% of the average. Use of this assay to screen for RNA polymerase inhibitors from chemical libraries and natural products led to the identification of DNA intercalators (known to inhibit RNA polymerase activity), rifampicin (a known inhibitors of RNA polymerase), and several derivatives of rifampicin from Actinomycetes extracts. Therefore this assay can be reliably utilized to detect novel inhibitors of bacterial RNA polymerase. 

Taking together, DNA polymerase structural data indicate a high degree of shape complementary between the active sites of the enzymes and the nucleotide substrates, suggesting that geometrical constraints are at least one cause of DNA polymerase fidelity. 

The 3 -+5 exonuclease activity plays an important role in polymerization in proof reading the base pair formed at each polymerization step. The enzyme checks the nature of each base-paired primer terminus before the polymerase proceeds to add the next nucleotide to the primer. It thus supplements the capacity of the polymerase to match the incoming nucleotide substrate to the template. A mismatched terminal nucleotide on the primer activates a site on the enzyme which results in the hydrolysis of the phosphodiester bond and the removal of the mismatched residue. The function of this 3 - 5 exonuclease activity is therefore to recognize and cleave incorrectly or non-base paired residues at the 3 -end of DNA chains. It will therefore degrade single stranded DNA and frayed or non-base paired residues at the ends of duplex DNA molecules provided they terminate in a 3 -hydroxyl group. 

Fig. 5.15. Strategy for the selection of a phage-displayed polymerase (Pol) [68]. The primertemplate substrate is derivatized with a maleimide reagent and one nucleotide substrate is derivatized with biotin (Bt).The phage is labeled by the product via the maleimide and subsequently captured with immobilised streptavidin (Sv).

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