Phosphoryl Group Transfer — Phosphatases

A/B Phosphatases Phosphoryl group transfer from a phosphoric monoester to water as an acceptor molecule. (Phosphoric monoesters are cleaved hydrolytically). 3.1.3. 3.6.1. Phosphoric ester hydrolases Hydrolases acting on acid anhydrides in phosphorous-containing anhydrides... 

Phosphoryl group transfer reactions add or remove phosphoryl groups to or from cellular metabolites and macromolecules, and play a major role in biochemistry. Phosphoryl transfer is the most common enzymatic function coded by the yeast genome and, in addition to its importance in intermediary metabolism (see Chapter 5), the reaction is catalysed by a large number of central regulatory enzymes that are often part of signalling cascades, such as protein kinases, protein phosphatases, ATPases and GTPases. 

Mechanistically related enzymes that are ubiquitous in their requirement for a metal ion are those that catalyze phosphoryl group transfer reactions. These enzymes include the (phospho)kinases and phosphatases, which catalyze the transfer of a phosphoryl group (PO3) from the y-position of a nucleotide to an acceptor molecule (the phosphatases are a special case wherein the acceptor is the solvent water) nucleotidyltransferases and nucleases, which catalyze the transfer of a nucleoside phosphodiester to an acceptor molecule (the nucleases and phosphodiesterases are special cases wherein the acceptor is the solvent water) and phosphomutases. In all of these cases, the mechanism of phosphoryl group transfer can occur through one of several postulated pathways. The possible mechanistic extremes are those described as dissociative, analogous to an Sn 1 reaction. 

Historically, the protein phosphatases were classified into two groups according to their substrate specificity tyrosine phosphatases and serine/threonine protein phosphatases. The latter are metalloenzymes we will refer to these as metallophospha-tases, a superfamily of enzymes that includes the serine/threonine protein phosphatases, purple acid phosphatases, and other enzymes capable of phosphoryl group transfer reactions such as bacteriophage A protein phosphatase [17, 18]. 

Phosphates of pharmaceutical interest are often monoesters (Sect. 9.3), and the enzymes that are able to hydrolyze them include alkaline and acid phosphatases. Alkaline phosphatase (alkaline phosphomonoesterase, EC 3.1.3.1) is a nonspecific esterase of phosphoric monoesters with an optimal pH for catalysis of ca. 8 [140], In the presence of a phosphate acceptor such as 2-aminoethanol, the enzyme also catalyzes a transphosphorylation reaction involving transfer of the phosphoryl group to the alcohol. Alkaline phosphatase is bound extracellularly to membranes and is widely distributed, in particular in the pancreas, liver, bile, placenta, and osteoplasts. Its specific functions in mammals remain poorly understood, but it seems to play an important role in modulation by osteoplasts of bone mineralization. 

FIGURE 15-14 Protein phosphorylation and dephosphorylation. Protein kinases transfer a phosphoryl group from ATP to a Ser, Thr, or Tyr residue in a protein. Protein phosphatases remove the phosphoryl group as P . 

Nucleophilic displacements on phosphorus (Table 10-1, reaction type 1C) are involved in virtually every aspect of cellular energetics and in many aspects of biosynthesis. One large group of such enzymes are phosphotransferases, which transfer phospho (also called phosphono or, traditionally in biochemistry, phosphoryl) groups from one nucleophilic center to another. When transfer is to water the enzymes are called phosphatases, and when from one group in a molecule to another in the same molecule, mutases. 

Studies on the transferase action of milk and intestinal phosphatases have shown that compounds such as glucose, glycerol, and propanediol can accept a phosphoryl residue from a wide variety of donors (IBS). The overall reaction is therefore transfer of a phosphoryl group from a donor of type (II) where X is F, 0, S, or N and R is H or an alkyl substituent, etc., or may even be absent, to an acceptor of type R —OH... 

As noted earlier, the binding of Mg2+ to ATP results in activation towards hydrolysis of the terminal phosphoryl group. Hydrolysis and transfer of phosphate may involve transfer of the phosphoryl group to the enzyme, followed by transfer to water or an acceptor molecule. Enzymes such as alkaline phosphatase and fructose-1,6-biphosphatase which require Mg2+ and Zn2+ will be considered in Section 62.1.4. 

Examination of the first partial reaction reveals that the mutase functions as a phosphatase—it converts 2,3-bisphosphoglycerate into 2-phosphoglycerate. However, the phosphoryl group remains linked to the enzyme. This phosphoryl group is then transferred to 3-phosphoglycerate to reform 2,3-bisphosphoglycerate. 

Table 2 lists phosphatases for which the stereochemical outcome has been determined by using chiral phosphomonoester substrates. In such experiments the phosphoryl group is made chiral using lfiO, 170, and lsO, or, a phosphorothioate substrate is used that has a sulfur atom and two isotopes of oxygen in the nonbridging positions. Summaries of results for additional enzymes that catalyze phosphoryl transfer are available in other reviews.20,76,77... 

Fig. 17 The two typical reaction mechanisms employed by phosphatases. In (a), the phos-phoryl group is transferred directly to a water molecule, which is typically bound to one or two metal ions if the substrate is made chiral at phosphorus, the stereochemical outcome is inversion. In (b), the phosphoryl group is first transferred to an enzymatic residue. In a subsequent step the phosphoenzyme intermediate is hydrolyzed. Since each step occurs with inversion of configuration at phosphorus, the net outcome is retention. Pi inorganic phosphate.

The use of a chiral [160,170,180]phosphoryl group to study the steric course of phosphoryl transfer reactions was developed mainly in two laboratories, initially in Knowles group and more recently in Lowe s group. The key step in Knowles approach is the reaction catalyzed by K coli alkaline phosphatase. This... 

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