In-line attack

It is generally assumed that hydrolysis of the y-phosphate bond proceeds via an Sn2 mechanism, as shown in Fig. 5.17. The hydrolysis proceeds by an in-line attack of a water molecule on the y-phosphate, in which the GDP residue is displaced from the y-phosphate. 

Fig. 5.17. In-line attack in GTP hydrolysis. Hydrolysis of GTP takes place via an in-tine attack of a water molecnle at the y-phosphate. The reaction passes through a pentavalent transition state in which the ligands of the y-phosphate adopt a trigonal bipyramidal configuration. The mechanism by which the water molecule is activated for the attack on the y-phosphate is not shown in the figure. Possible mechanisms are presented in Fig. 5.18.

The catalytic center is formed by residues from both lobes. Sequence comparisons, mutation experiments and biochemical studies indicate an essential fimction in catalysis of phosphate transfer for the conserved amino acids Lys72, Aspl66 and Aspl84 (numbering of PKA). However, the catalytic mechanism of phosphate transfer is not definitely established. It is generally assumed that Aspl66, which is invariant in all protein kinases, serves as a catalytic base for activation of the Ser/Thr hydroxyl and that the reaction takes place by an in-line attack of the Ser-OH at the y-phosphate. 

Fig. 8.17. Mechanism of hydrolysis of phosphotyrosine residues by tyrosine phosphatases. Cleavage of phosphate from phosphotyrosine residues takes place by an in-line attack of a nucleophilic cysteine thiolate of the tyrosine phosphatase at the phosphate of the phosphotyrosine residue. The negative charge on the thiolate is stabilized by the positive charge of a conserved Arg residue. In the course of the reaction, an enzyme-Cys-phosphate intermediate is formed, which is hydrolytically cleaved to phosphate and enzyme-Cys-SH. The figure shows selected interactions. Other interactions in the active center involved in substrate binding and catalysis are not shown. According to Tainer and Russel, (1994). R substrate protein.

In the high resolution crystal structure of the GTP form of Ras protein, a tightly bound water molecule is visible located in an optimal position for nucleophilic attack on the y-phosphate (Wittinghofer et al., 1993). The water molecule is fixed in a defined position by H-bridges with GIn61 and Hir35. As described in 5.4.4 for the a-subunits of the heterotrimeric G-proteins, GTP hydrolysis takes place by an in-line attack of the nucleophilic water molecule on the y-phosphate, for which a pentagonal, bipyramidal transition state is postulated. 

The resulting HO probably carries out a direct in-line attack as shown in Fig. 12-29. Mutants such as E43D, E43Q, and E43S have greatly decreased activity,829 in... 

Figure 12-29 Drawing showing the hydrogen-bonding interactions between the guanidinium ions of arginines 35 and 87 of the micrococcal (staphylococcal) nuclease with the 5 -phosphate of the inhibitor thymidine 3, 5 -diphosphate in the complex of E + I + Ca2+. A possible mechanism is illustrated. A hydroxyl ion bound to Ca2+ carries out an in-line attack on the phosphorus. See Libson et al.S26...

Restriction endonuclease EcoRI is able to cut a chain in dsDNA which has a chiral phophorothioate group at the specific cleavage site.838 Tire reaction occurs with inversion of configuration at phosphorus, suggesting direct in-line attack by a hydroxyl ion generated from H20. 

The methyl ester was crystallized and its absolute stereochemistry was determined by x-ray diffraction to be as in equation 8.37. This product corresponds to an in-line attack. When incubated with ribonuclease in aqueous solution, the methyl ester re-forms the original cyclic phosphorothioate (structure 8.36). This result is expected from the principle of microscopic reversibility, since the forward and reverse reactions must go through the same transition state. But it does show directly that the cyclization step involves an in-line attack an adjacent attack of the ribose hydroxyl in the cyclization of the methyl ester as in the right-hand structure 8.38 would give the enantiomer of structure 8.36. 

Folding into the active conformation requires a sharp bend between helix 2 and helix 3 to orient the two helical domains in an antiparallel fashion and to enable Loop A to interact with Loop B (Figure 5.2.7). Catalysis is achieved by specific positioning of the phosphodiester bond to be processed within the ribozyme architecture, such that nucleophilic in line attack of the 2 -OH at the phosphorus atom in an SN2-like mechanism becomes possible (Figure 5.2.6). 

Two alternative mechanisms of ATP hydrolysis that appear both steri-cally and chemically reasonable can be suggested. The first possibility would be a direct in-line attack by a H2O molecule on the y-phosphate of ATP, with Asp-206 acting as a proton acceptor. The plausibility of this alternative was initially suggested by the comparison of the actin and HSC70 ATPase fragment structures, and more specifically by superposition of a CaATP nucleotide, with the conformation observed in actin, on the ATPase fragment model (Flaherty et ai, 1991). An HgO molecule appears to intercalate between the carboxyl of Asp-206 and the hypothet-... 

Hydrolysis of GTP proceeds by an (Sn2) in-line attack of a water molecule on the y-phosphate of GTP. A Gly, a Gin, and an Arg residue are involved in catalysis. Mutations at any of these sites largely abolish GTP hydrolysis and stabilize the active GTP-bound state.A constitutive, unregulated, permanent activity of the GTP-bound state can be harmful, as indicated by the consequences of mutations at these sites in Gj-a (Gg-a is the common stimulatory G protein, see Chapter 5). Mutated Gj-a is found in human endocrine tumours b92 nd comparable mutations in Ras are oncogenic. ... 

Since concerted bond breaking and leaving group protonation was found to be considerably favored over a stepwise mechanism in the first part of the reaction, the analogous concerted pathway was also modeled here. Simulation of the first step showed that the protein environment cannot stabilize a negative ligand in the active site outside the phosphate binding loop, which would also be the case for a stepwise proton transfer to Asp 129 and a subsequent in-line attack of a hydroxide ion. 

To explore the divalent and monovalent metal ion binding modes (Figure 1) and their relation to formation of catalytically active, in-line attack conformations in both the neutral reactant and activated precursor (deprotonated 2 OH nucleophile) states, we set up the following series of simulations ... 

A bridging Mg2+ ion maintains rigid coordination patterns that stabilize in-line attack conformations... 

Distances and angles (Figure 1) are in angstrom and degrees, respectively. The average values, denoted as (...), are obtained by averaging over all snapshots in the cluster. R is the in-line attack distance (C17 02 to C1.1 P). 6 is the in-line attack angle (between C17 02, C1.1 P, and C1.1 05 ). N is the number of ions with at least one coordination to any one of the four coordination sites. CN is the total coordination number of all ions with at least one coordination to any one of the four coordination sites. NB is the number of ions which coordinate to at least two of the four coordination sites. 

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