Phosphorylating enzyme

FIGURE 15.2 Enzymes regulated by covalent modification are called interconvertible enzymes. The enzymes protein kinase and protein phosphatase, in the example shown here) catalyzing the conversion of the interconvertible enzyme between its two forms are called converter enzymes. In this example, the free enzyme form is catalytically active, whereas the phosphoryl-enzyme form represents an inactive state. The —OH on the interconvertible enzyme represents an —OH group on a specific amino acid side chain in the protein (for example, a particular Ser residue) capable of accepting the phosphoryl group. 

Interestingly, however, the mechanisms of the two phosphate hydrolysis reactions in steps 9 and 11 are not the same. In step 9, water is the nucleophile, but in the glucose 6-phosphate reaction of step 11, a histidine residue on the enzyme attacks phosphorus, giving a phosphoryl enzyme intermediate that subsequently reacts with water. 

The primary site of action of OPs is AChE, with which they interact as suicide substrates (see also Section 10.2.2 and Chapter 2, Figure 2.9). Similar to other B-type esterases, AChE has a reactive serine residue located at its active site, and the serine hydroxyl is phosphorylated by organophosphates. Phosphorylation causes loss of AChE activity and, at best, the phosphorylated enzyme reactivates only slowly. The rate of reactivation of the phosphorylated enzyme depends on the nature of the X groups, being relatively rapid with methoxy groups (tso 1-2 h), but slower with larger... 

Reactivators of phosphorylated ChE. Pyridine aldoxime methiodide (PAM) and related compounds are the best known. They reactivate the phosphorylated enzyme so long as aging has not occurred. They do not, however, reactivate the aged enzyme. ChE which has been phosphorylated by certain nerve gases ages rapidly ... 

Deoxy-3-fluoro-D-glucose (see Section 11,2), a weak substrate for yeast hexokinase, is phosphorylated enzymically - to give the 6-phosphate 588, which is transformed into 2-deoxy-2-fluoro-D-arabinose 5-phos-phate (589) by lead tetraacetate oxidation. 

Deoxy-5 -fluorothymidine (838) was prepared by Langen and Kowol-jj. 796,797 fpQjyj 5 -0-tosyl precursor by treatment with fluoride. Compound 838 cannot be phosphorylated enzymically owing to the lack of OH-5, but it inhibits the growth of carcinoma cells. This was explained as follows the thymidine 5 -monophosphate (thymidylate) kinase in carcinoma cells, catalyzing the transformation of thymidine 5 -monophosphate into the diphosphate, is inhibited by 838, thus preventing the synthesis of... 

The two substrate kinetics of the overall reaction catalyzed by the complex in permeabilized membranes showed classical ping-pong kinetics in accordance with a phosphorylated enzyme intermediate. The affinity constants for fructose and P-enolpyruvate were 8 and 25 /iM, respectively. 

Organophosphate and carbamate pesticides are potent inhibitors of the enzyme cholinesterase. The inhibition of cholinesterase activity by the pesticide leads to the formation of stable covalent intermediates such as phosphoryl-enzyme complexes, which makes the hydrolysis of the substrate very slow. Both organophosphorus and carbamate pesticides can react with AChE in the same manner because the acetylation of the serine residue at the catalytic center is analogous to phosphorylation and carbamylation. Carbamated enzyme can restore its catalytic activity more rapidly than phosphorylated enzyme [17,42], Kok and Hasirci [43] reported that the total anti-cholinesterase activity of binary pesticide mixtures was lower than the sum of the individual inhibition values. 

All isoforms of PKC are predominantly localized to the cytosol and, upon activation, undergo translocation to either plasma or nuclear membranes. However, newly synthesized PKCs are localized to the plasmalemma and are in an open conformation in which the auto inhibitory pseudosubstrate sequence is removed from the substrate binding domain. The maturation of PKC isoforms is effected by phosphoinositide-dependentkinase-I (PDK-I), which phosphorylates a conserved threonine residue in the activation loop of the catalytic (C4) domain [24]. This in turn permits the autophosphorylation of C-terminus threonine and serine residues in PKC, a step which is a prerequisite for catalytic activity (see also Chs 22 and 23). The phosphorylated enzyme is then released into the cytosol, where it is maintained in an inactive conformation by the bound pseudosubstrate. It was originally thought that 3-phosphoinositides such as PI(3,4)P2 and PI(3,4,5)P3 could directly activate PKCs. However, it now seems more likely that these lipids serve to activate PDK-1 (a frequent contaminant of PKC preparations). 

TABLE 23-3 Examples of proteins regulated by phosphorylation Enzymes involved in neurotransmitter biosynthesis Tyrosine hydroxylase Tryptophan hydroxylase Neurotransmitter receptors Adrenergic receptors Dopamine receptors Opioid receptors Glutamate receptors Many others... 

Another disadvantage of the first generation reactivators lies in their lacking of antidotic effect in respect to affections induced by soman, which causes fast aging of the phosphorylated enzyme. 

There are two different glucose-phosphorylating enzymes, hexokinase and glucokinase, which catalyse the reaction ... 

There are several different glucose transporters. There are two glucose phosphorylating enzymes glucokinase and hexokinase (Chapter 6). 

An additional phosphorylation (enzyme phosphatidylinositol-4-phosphate kinase 2.7.1.68) finally provides phosphaditylino-sitol-4,5-bisphosphate (PIP2, Ptdlns(4,5)P2). PIP2 is the precursor for the second messengers 2,3-diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (InsPa, IP3 see p. 367). 

Acyl-phosphates also display an unusual U -shaped pH profile for the rate of hydrolysis (see Fig. 1). This reflects the fact that acyl-phosphate compounds are susceptible to both acid- and base-catalyzed hydrolysis, and the shoulder at less acidic pH values reflects the slightly faster hydrolysis of the monoanion, compared to the dianion. Observation of a U -shape pH-rate profile for a previously uncharacterized phosphoryl-enzyme intermediate supports the inference that an acyl-phosphate has formed. 

Figure 1. Reaction cycle showing how ATP hydrolysis and transient formation of a phosphoryl-enzyme intermediate are linked to the vectorial transport of calcium in the sarcoplasmic reticulum.

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