Stereochemistry phosphoryl transfer

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

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...

Figure 6.52 Mechanistic possibilities for transfer of a monophosphate, (a) 5 1 (P) involving a solvent-equilibrated metaphosphate intermediate. Protonation states are not shown, but different fonts for the phosphorus oxygens, representing different oxygen isotopes, indicate stereochemistry. (b) Canonical structures for metaphosphate and alkyl phosphate dianion, (c) 5n2(P) involving an intermediate which can pseudorotate, (d) Exploded 5Nl(P)-like >Sn2(P) transition states, and cartoon of calculated movements of phosphorus and non-bridge oxygens during phosphoryl transfer through such a transition state, where the leaving to nucleophile atom distance is constant.

Mg " and soft metals such as being used. The ability to replace a phosphate oxygen by sulfur, in combination with the use of soft metals by phosphoryl-transferring enzymes, provides an important probe of stereochemistry and mechanism. If sulfur is incorporated at a site coordinating to (or a hydrogen bond donor such as lysine), catalysis is gravely impaired, whereas if the coordination is to Zn " there is little effect. 

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... 

The structure and function of phosphoproteins has provoked much interest in the past year. The application of n.m.r. to the study of protein structure and function has been reviewed while the stereochemistry of enzymic phosphoryl transfer and the enzymology of kinases have been described in recent volumes of Methods in Enzymology. The identification of the sites of phosphorylation on phosphoproteins is an important prerequisite in attempting the elucidation of the functions of these compounds and methods have been described for the detection of 0-phosphoserine and 0-phosphotyrosine residues in proteins. The enzymic phosphorylation and dephosphorylation of tyrosine residues in proteins has been investigated, and a simple synthesis of O-phospho-L-tyrosine from the amino acid and a phosphoric oxide/orthophosphoric acid mixture has been described. ... 

One particularly fascinating class of pentacoordinate phosphorus compounds is the class of chiral spirophosphoranes with amino acid residues as chiral chelate ligands since they can serve as important structural features relevant to chiral phosphoryl transfer pathways. Therefore, their synthesis and stereochemistry were recently widely explored." ... 

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. 

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. 

Use of Chiral Thiophosphates and the Stereochemistry of Enzymatic Phosphoryl Transfer... 

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... 

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+. 

The transaldolase (EC 2.2.1.2) is an ubiquitous enzyme that is involved in the pentose phosphate pathway of carbohydrate metabolism. The class I lyase, which has been cloned from human [382] and microbial sources [383], transfers a dihydroxyacetone unit between several phosphorylated metabolites. Although yeast transaldolase is commercially available and several unphosphorylated aldehydes have been shown to be able to replace the acceptor component, preparative utilization has mostly been limited to microscale studies [384,385] because of the high enzyme costs and because of the fact that the equilibria usually are close to unity. Also, the stereochemistry of transaldolase products (e.g. 38, 40) [386] matches that of the products from the FruA-type DHAP aldolase which are more effortlessly obtained. 

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. 

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