Nucleotide incorporation pathway steps

Fig. 1. Kinetic pathway of nucleotide incorporation. The various complexes are indicated as mentioned in the text. kpol, the rate constant of the rate-limiting step, is indicated.

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... 

Another group of inhibitors prevents nucleotide biosynthesis indirectly by depleting the level of intracellular tetrahydrofolate derivatives. Sulfonamides are structural analogs of p-aminobenzoic acid (fig. 23.19), and they competitively inhibit the bacterial biosynthesis of folic acid at a step in which p-aminobenzoic acid is incorporated into folic acid. Sulfonamides are widely used in medicine because they inhibit growth of many bacteria. When cultures of susceptible bacteria are treated with sulfonamides, they accumulate 4-carboxamide-5-aminoimidazole in the medium, because of a lack of 10-formyltetrahydrofolate for the penultimate step in the pathway to IMP (see fig. 23.10). Methotrexate, and a number of related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. These inhibitors are structural analogs of folic acid (see fig. 23.19) and bind at the catalytic site of dihydrofolate reductase, an enzyme catalyzing one of the steps in the cycle of reactions involved in thymidylate synthesis (see fig. 23.16). These inhibitors therefore prevent synthesis of thymidylate in replicating... 

The evolutionarily conserved Moco biosynthetic pathway consists of three steps (Scheme 1) (1) the conversion of a guanosine derivative (probably guanosine triphosphate) into sulfur-free Precursor Z via insertion of C-8 between the ribose C-2 and C-3 atoms, (2) sulfurization and transformation of Precursor Z into MPT, catalyzed by MPT-synthase, and (3) metal incorporation through chelation of the dithiolene moiety. Additional steps are involved in the attachment of a nucleotide to generate the dinucleotide forms found... 

A theory proposing that the effect of steroids is to stimulate amino acid activation and, as a result, protein synthesis is not without objections. If the primary effect of steroid hormones is to activate amino acids, the rate of synthesis of all proteins should be increased in the target organ. In contrast, steroids stimulate the biosynthesis of highly specific proteins, and therefore it seems more logical to assume that the steroids act at a step of the biosynthetic pathway that determines the specificity of the proteins synthesized. Such a mechanism could involve transcription of messenger RNA on DNA template. The rate of incorporation of labeled nucleotide triphosphate into the rapidly labeled nuclear RNA by the methods described by Weiss is markedly decreased in castrated animals. 

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