In-line displacement

The stereochemical mechanism of 3 processing and strand transfer has been investigated using DNA substrates that incorporate phosphorothioate link-ages [8]. For both reactions, the introduced chiral centers are inverted in the products, implying that the reactions occur via a one-step in-line displacement mechanism rather than via a covalent intermediate. 

The chemical reactivities of groups in the apical and equatorial positions of pentacovalent intermediates are different.664 In particular, elimination of a nucleophilic group to form a tetrahedral phosphate is easier from an apical position than from an equatorial position. For the in-line displacement of Eq. 12-27 elimination of RO should be easy. However an adjacent attack would leave - OR in an equatorial position. Before it could be eliminated, the intermediate would probably have to undergo a permutational rearrangement by which -OR would be transferred from an equatorial to an apical position. 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

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...

For the chiral phenylphospholanium salts, modeling (13) shows, in agreement with the initial suggestion of MarsiTT4), that steric control in the ground state is important in accounting for the decreased amount of inversion at phosphorus for 8 compared to that for In 8, in-line displacement of the methoxy... 

Each postulates a different nucleophile to carry out the attack on the phosphorus. In either case, each reaction takes place by an in-line displacement path ... 

Cleavage Is by In-Line Displacement of 3 -Oxygen from Phosphorus by Magnesium-Activated Water... 

Interestingly, Equation (8.75) and the irreversible, single catalytic site. Ter Ter kinetic scheme may also be applicable for the steady state kinetic analysis of the overall conversion of two molecules of ATP to Ap4A and pyrophosphate, catalysed by LysU with the assistance of the third substrate lysine-Mg + (Figure 8.11, Table 8.1). The combination of ATP, the hrst nucleotide substrate, with lysine-Mg to give intermediate lysyl adenylate represents the formation of a transient non-covalent enzyme-intermediate complex. Pyrophosphate product now dissociates from the catalytic site allowing for ATP, the second nucleotide substrate, to enter in its place and form Ap4A by in-line displacement of lysine from the adenylate... 

Metal species have often proved to be essential components in many DNA and RNA cleavage systems. Effective metal ion-centred catalytic systems for the hydrolysis of phospho-diester bonds have been proposed which employ metal ions both to deliver a hydroxide ion in a direct in-line displacement and to complex the incipient alkoxide leaving group. This type... 

Inorganic chiral thiophosphate analysis of fructose bisphosphatase indicates overall inversion of stereochemistry (Domanico et ai, 1986). To distinguish between direct in-line displacement and the intermediacy of an acyl phosphate, a single-turnover experiment was performed. Isotopic equilibration of the putative enzyme-CO 2 with labelled water through multiple turnovers leads to label incorporation. A subsequent single turnover experiment with one equivalent of substrate in unlabelled water must lead to label... 

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