Phosphatases phosphoryl group transfer kinases

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. 

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 . 

Figure 17-1 Universal regulation by protein phosphorylation. Protein phosphorylation requires the coordinated actions of protein kinases, which transfer a phosphoryl group to a target protein, and protein phosphatases, which remove it via hydrolysis. Phosphorylation of a target protein can change its biological activity in many ways including enzymatic activity, intracellular localization, and its ability to interact with other macromolecules such as DNA, RNA, and proteins. The most common amino acids which are phosphorylated in eukaryotic organisms are serine, threonine, and tyrosine. Phosphorylation on histidine with subsequent phosphoryl transfer to aspartic acid represents a coitunon modification in prokaryotic two-component signal transduction s)rstems (see Figure 17-15A).

In contrast to tyrosine kinases, Tyrosine phosphatases (PTPs) are enzymes which act on phosphorylated proteins and catalyze the transfer of a phosphate group from a tyrosine residue to a water molecule, generating orthophosphates in a process which is referred to as dephosphorylation. PTPs are involved in many cellular signal transduction pathways. 

Figure 2. Mechanism of PDH. The three different subunits of the PDH complex in the mitochondrial matrix (E, pyruvate decarboxylase E2, dihydrolipoamide acyltrans-ferase Ej, dihydrolipoamide dehydrogenase) catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. E, decarboxylates pyruvate and transfers the acetyl-group to lipoamide. Lipoamide is linked to the group of a lysine residue to E2 to form a flexible chain which rotates between the active sites of E, E2, and E3. E2 then transfers the acetyl-group from lipoamide to CoASH leaving the lipoamide in the reduced form. This in turn is oxidized by E3, which is an NAD-dependent (low potential) flavoprotein, completing the catalytic cycle. PDH activity is controlled in two ways by product inhibition by NADH and acetyl-CoA formed from pyruvate (or by P-oxidation), and by inactivation by phosphorylation of Ej by a specific ATP-de-pendent protein kinase associated with the complex, or activation by dephosphorylation by a specific phosphoprotein phosphatase. The phosphatase is activated by increases in the concentration of Ca in the matrix. The combination of insulin with its cell surface receptor activates PDH by activating the phosphatase by an unknown mechanism.

FIGURE 24-2 Tyrosine phosphorylation and dephosphorylation. Protein tyrosine kinases (PTK) catalyze the transfer of the y-phosphate group from ATP to the hydroxyl group of tyrosine residues, whereas protein tyrosine phosphatases (PTP) remove the phosphate group from phosphotyro-sine. R, protein. 

Protein phosphorylation dephosphorylation regulates the activity of enzymic and nonenzymic proteins in eukaryotic cells. Ten per cent or more of all proteins in a cell are modified in that way. The phosphates are transferred from ATP and esterified with hydroxyl groups of serine, threonine, or tyrosine residues. They are removed and transferred to water by phosphatases. There are at least about 2000 kinases and about 1000 phosphatases to carry out these reactions (Fig. 7.1). ... 

In some kinases, such as nucleoside diphosphate kinase, " an intermediate step is the phosphoryl transfer to a group belonging to the enzyme, as happens in ATPase and as was discussed in detail for alkaline phosphatase (Section V.B). In other kinases the phosphoryl transfer occurs directly from the donor to the acceptor in a ternary complex of the enzyme with the two substrates.Often metal ions like magnesium or manganese are needed. These ions interact with the terminal oxygen of the ATP molecule, thus facilitating the nucleophilic attack by the acceptor. The metal ion is often associated with the enzyme. For mechanistic schemes, see the proposed mechanism of action of alkaline phosphatase, especially when a phosphoryl enzyme intermediate is involved. 

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