Phosphoryl transfer reactions enzyme active sites

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... 

Metal Ion Effects. The metal ion effects on the acid-catalyzed hydrolysis of PPS also were examined by Benkovic and Hevey (5). However, they observed that in water near pH 3, the rate enhancement in the presence of an excess of metal ion was at most only threefold (Mg2+, Ca2+, Al3+) and in some cases (Zn2+, Co2+, Cu2+) the rate was actually retarded. We thought that the substrate PPS and Mg2+ ion should be hydrated heavily in water so that their complexa-tion for rate enhancement is weak. If, however, the hydrolysis is carried out in a solvent of low water content, such complexation would not occur, and therefore, the rate enhancement might be more pronounced. This possibility appears to be supported by the fact that the active sites of many enzymes are hydrophobic. Of course, there is a possibility that the S—O fission may not require metal ion activation. In this connection, it is interesting to note that in biological phosphoryl-transfer reactions the enzymes generally require divalent metal ions for activity (7, 8, 9), but such metal ion dependency appears to be less important for sulfate-transfer enzymes. For example, many phosphatases require metal ions, but no sulfatase is known to be metal... 

A theoretical study based on MP2/6-31+G(d,p) and HF/6-31G(d) ab initio quantum mechanical calculations coupled with Langevin dipoles (LD) and polarised continuum (PCM) solvation models have been carried out by Florian and Warshel [387] to achieve a first systematic study of the free energy surfaces for the hydrolysis of methylphosphate in aqueous solution. The important biological implication of this work is the fact that since the energetics of both the associative and the dissociative mechanics are not too different, the active sites of enzymes can select either mechanism depending on the particular electrostatic environment. This conclusion basically means that both mechanisms should be considered, and this fact seems to contradict some previous studies which have focused on phosphoryl transfer reactions. 

Structural studies as well as sequence comparisons among polymerases strongly suggest the hypothesis that the phosphoryl transfer reaction of all polymerases is catalyzed by a two metal ion mechanism that was originally proposed by analogy to the well studied two metal mechanism in the 3 exonuclease reaction (14). It is perhaps of interest to note that such a mechanism, which involves only the properties of two correctly positioned divalent metal ions, could easily be used by an enzyme made entirely of RNA and thus could function in an all RNA world. The fidelity of DNA synthesis appears to arise from two sources. First, enforced Watson-Crick interactions at the polymerase active site increases the accuracy of the incorporation step (9,13). Second, there is a competitive editing at the 3 exonuclease active site that removes misincorporated nucleotide (3,5). When nucleotides are... 

As has been already mentioned, theoretical investigations suggest mechanisms of phosphoryl transfer reactions that involve pentacoordinated phosphorus atom that can be a trigonal bypiramidal intermediate or transition state. However, recent experimental work has outlined the involvement of higher coordinate forms of phosphorus, particularly the ease of formation of hexacoordinated phosphoranes. These species can be formed by utilising residues at active sites of phosphoryl transfer enzymes to enter into donor interactions at the phosphorus atom and as a consequence cooperate in nucleophilic attack. To prove this assumption, biorelevant phosphoranes have been synthesized and studies of their behaviour in active sites of enzymes have been described. As representative analogues of a transition state in phosphoryl transfer reaction some phosphorus-atrane... 

It is generally claimed that phosphoryl transfer may follow basically two pathways (see O Fig. 5.1). In the dissociative mechanism a trigonal metaphosphate intermediate is formed (Xu and Guo 2008), while the associative mechanism involves a relatively stable, trigonal bipyra-midal intermediate (Lahiri et al. 2003). Note that an intermediate refers to a local energy minimum on the reaction path. However, a third option has to be mentioned, too, this is the classical Sn2 mechanism with a trigonal bipyramidal transition state, referring to a maximum on the reaction path (Bernardi et al. 2002). The preferred pathway is determined by the nature of the phosphorus electrophile, the nucleophile, and the reaction medium (solvent or enzyme active site). Earlier computer simulations indicate that associative and dissociative mechanisms are similarly favored in the aqueous phase (Floridn and Warshel 1998), and also calculations for different enzymes support either dissociative or associative pathways depending on a variety of factors (Klahn et al. 2006) (O Fig. 30-9). 

Although this reaction is fully reversible, the relatively high [ATP]/[ADP] ratio in cells normally drives the reaction to the right, with the net formation of NTPs and dNTPs. The enzyme actually catalyzes a two-step phosphoryl transfer, which is a classic case of a double-displacement (Ping-Pong) mechanism (Fig. 13-12 see also Fig. 6-13b). First, phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme... 

Most kinases transfer chiral phospho groups with inversion and fail to catalyze partial exchange reactions that would indicate phosphoenzyme intermediates. However, nucleoside diphosphate kinase contains an active site histidine which is phosphorylated to form a phosphoenzyme.869 The enzyme catalyzes phosphorylation of nucleoside diphosphates other than ADP by a nucleotide triphosphate, usually ATP. 

Available evidence (14,15) favors the pathway for pyruvate kinase by way of phosphorylation of pyruvate enol. Furthermore, J. Knowles and his coworkers (16,17), using chiral thiophosphates and chiral (160,170,180) phosphate have shown that pyruvate kinase transfers phosphate from phosphoenolpyruvate to ADP with stereochemical inversion at phosphorus. Since monomeric metaphosphate is presumably planar, a chemical reaction by way of that ion should proceed with racemization. In the active site of an enzyme, however, all components might be held so rigidly that racemization need not occur. Furthermore, no information is yet available on the detailed mechanism of reactions catalyzed by cytidine synthetase our own experiments, designed to distinguish among the mechanisms here discussed, are as yet incomplete. 

The enzyme can also catalyze the transfer of the terminal phosphoryl of ATP to water i.e., it acts as an ATPase but at a rate 5 x 106 times slower than the above reaction. The basic and nucleophilic properties of water versus the C-6 hydroxyl of glucose are sufficiently similar to suggest no marked differences in rate. Therefore, the explanation of the rate difference is that glucose induces a conformational change that establishes the correct active-site geometry in the enzyme, whereas a water molecule is too small to do so. 

Figure 1 Chemical mechanism of DNA polymerase and 3 -5 exonuclease, (a) DNA polymerase reaction. The enzyme chelates two metal Ions using three aspartic acid residues (only two are shown). Metal ion A abstracts the 3 hydroxyl proton of the primer terminus to generate a nucleophile that attacks the a-phosphate of an incoming dNTP substrate. The phosphoryl transfer results In production of a pyrophosphate leaving group, which is stabilized by metal Ion B. (b) The 3 -5 exonuclease proofreading activity is located in a site that is distinct from the polymerase site yet it uses two-metal-ion chemistry similar to DNA synthesis. The reaction type is hydrolysis in which metal ion A activates water to form the hydroxy anion nucleophile. Nucleophile attack on the phosphate of the mismatched nucleotide releases it as dNMP (dGMP in the case shown).

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