Phosphoryl transfer reactions stereochemistry

Triply labeled phosphate has been employed to elucidate the steric course of a number of phosphoryl transfer reactions the topic has been reviewed 165>166>, and we shall here present only one example, concerned with the stereochemistry of cycliza-tion of ADP to cAMP 167,168) and the reverse reaction 169,170). 

Table 2 Stereochemistry of some enzyme-catalyzed phosphoryl transfer reactions...

Enzymatic phosphoryl transfer reactions are ubiquitous in nature and play significant roles in ATP hydrolysis and protein phosphorylation processess. As previously described, pentacoordinate phosphorus species have been assumed as transient intermediates or transition states in these pathways and their structural and electronic properties are undoubtedly related to the outcome of the process. Therefore, to aid understanding of the phosphorus-catalyzed biological routes, many model pentacoordinated phosphoranes have been synthesized. While most studies have focused on aspects of apicophilicity, anti-apicophilicity or Berry pseudorotation, there have been limited investigations on the stereochemistry of pentacoordinated spirophosphoranes with a chiral phosphorus atom. In the past year, much attention has been paid to the synthesis and determination of absolute configuration of several chiral pentacoordinate spirophosphoranes derived from D- and L-aminoacids. Some significant achievements in this area will be discussed in this section. 

The use of thiophosphate analogs of biophosphates in studying stereochemical problems was first introduced by Eckstein (1975) and subsequently widely applied to various problems. To illustrate the use of chiral thiophosphates in stereochemistry, consider the phosphoryl transfer reaction catalyzed by hexokinase (Scheme 1). Three types of problems can be studied by... 

Tabic 8.1 Stereochemistry of enzyme-reactions" catalyzed phosphoryl transfer ... 

The PAP-catalyzed reaction occurs with inversion of stereochemistry at phosphorus, supporting the direct phosphoryl transfer from substrate to a metal-coordinated water or hydroxide nucleophile/ Inspection of the active site (Figure 9) reveals two candidates, a monodentate hydroxo/aquo moiety coordinated to the ferric ion, and a bridging hydroxide. Debate surrounds the identity of the nucleophile and of the substrate-binding mode three mechanistic proposals are summarized in Scheme... 

Two new techniques have been introduced that are especially useful when no stable intermediate can be isolated. Thiophosphates or phosphates with isotopically labeled oxygen atoms are chiral and can thus be us to probe the stereochemistry of the phosphoryl transfer event (Eckstein, 1979 Knowles, 1980). One in-line displacement leads to an inversion of configuration on the phosphorus atom. If one covalent intermediate is involved, retention of configuration should result from the double-displacement reaction. Using this technique, the existence of the acetate kinase acyl phosphate intermediate has been challenged (Knowles, 1980), whereas others (Spector, 1980) have interpreted the same results as being indicative of a triple-displacement mechanism. 

In animals and in many bacteria, PEP is formed by decarboxylation of oxaloacetate. In this reaction, which is catalyzed by PEP carboxykinase (PEPCK), a molecule of GTP, ATP, or inosine triphosphate captures and phosphorylates the enolate anion generated by the decarboxylation (Eq. 13-46).252 The stereochemistry is such that C02 departs from the si face of the forming enol.253 The phospho group is transferred from GTP with inversion at the phosphorus atom 254 The enzyme requires a divalent metal ion, preferably Mn2+. 

Mg2+ is associated with a large number of enzymes involving the hydrolysis and transfer of phosphates. The MgATP complex serves as the substrate in many cases. As noted in Section 62.1.2.2.2, the interaction of Mg2+ with the ATP enhances the transfer (to a substrate or water) of the terminal phosphoryl group. The results of many studies with model compounds lead to the postulate of an SN2 mechanism for this reaction.125 Associative pathways allow greater control of the stereochemistry of the substitution, and the rates of such processes are accelerated more effectively by metal ions. 

A sensitive probe applied to understand the nature of the reaction mechanism of group transfer is the stereochemistry of the overall reaction. The reaction at a phosphoryl center normally is a degenerate question, since a monosubstituted phosphate ester or anhydride is proprochiral at the phosphate center. Phosphate centers at a diester or disubstituted anhydride are prochiral. Two related methods to analyze the stereochemistry at a phosphate center have been developed by the generation of chirality at the phosphorus center. The first approach was developed by Usher et al. (24) and gave rise to the formation of isotopi-cally chiral [ 0, 0]thiophosphate esters and anhydrides (I). Isotopically chiral [ 0, 0, 0]phosphates (II) have also been synthesized and the absolute configurations determined. Two primary problems must first be addressed with respect to both of the methods that have been developed the synthesis of the isotopically pure chiral thiophosphates and phosphates and the analysis of the isotopic chirality of the products. An example of the chiral starting substrates, as developed for ATP, is schematically demonstrated. Ad = adenosine. 

As is known for other nucleoside kinases, the steady-state kinetics of the adenosine kinase reaction is complex owing to regulatory effects. Adenosine and AMP apparently bind at a regulatory site, where they modulate activity, as well as at the active site, where they act as substrates. These interactions complicate the kinetics, but a careful analysis shows that the basic kinetic pathway is sequential and involves the compulsory formation of ternary complexes (79). Thus, the kinetics is consistent with the stereochemistry and suggests that the phospho transfer is a direct, one-step displacement between substrates bound at the active site in a ternary complex. Complications introduced into the mechanistic analysis of this enzyme by the adventitious phosphorylation of the protein by ATP have been discussed elsewhere (7). 

These enzymes are not classified as nucleotidyltransferases, although they catalyze nucleotidyl group transfers in the course of activating the S -phosphoryl groups for the ligation process. The activation mechanism involves a covalent adenylyl-enzyme as an intermediate and a double displacement on of ATP (or NAD+). The chemical mechanism of the RNA ligase reaction is similar. The stereochemistry of these reactions is known for RNA ligase and is consistent with the mechanism as formulated above (81, 82). 

Stereochemistry is another powerful tool for determining the net reaction pathway of phosphatases and sulfatases. These enzymes catalyze the net transfer of a phosphoryl or sulfuryl group to water from a monoester, producing inorganic phosphate or sulfate. Inversion results when the reaction occurs in a single step (Scheme 2, pathway a). Phosphatases that transfer the phosphoryl group directly to water with inversion typically possess a binuclear metal center and the nucleophile is a metal-coordinated hydroxide. Examples of phosphatases that follow this mechanism are the purple acid phosphatases (PAPs) and the serine/threonine phosphatases (described in Sections 8.09.4.3 and 8.09.4.4.1). Net retention of stereochemistry occurs when a phosphorylated or sulfiirylated enzyme intermediate is on the catalytic pathway, which is hydrolyzed by the nucleophilic addition of water in a subsequent step (Scheme 2, pathway b). 

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