Enzyme-labile phosphate

The first study concerning the cell permeation of an inhibitor directed against the aldolase of T. hrucei using four different enzyme-labile phosphate protecting groups (10) has been presented. ... 

An exo-linker according to Fig. 10.1 must contain an enzyme labile group R, which is recognized and attacked by the biocatalyst Possible combinations could be phenylacetamide/penicillin amidase, ester/esterase, monosaccharide/glycosid-ase, phosphate/phosphatase, sulfate/sulfatase and peptides/peptidases [41]. The following systems have been worked out (Tab. 10.2). 

Figure 23.12. Bond Cleavage by PLP Enzymes. Pyridoxal phosphate enzymes lahilize one of three bonds at the a-carhon atom of an amino acid substrate. For example, bond a is labilized by aminotransferases, bond b by decarboxylases, and bond c by aldolases (such as threonine aldolases). PLP enzymes also catalyze reactions at the ()- and y-carbon atoms of amino acids. 

Hepatic glycogen synthase is similar to the muscle enzyme, although it is encoded by different genes. It is inactivated by phosphorylation and activated by dephosphorylation and may contain 12 mol of alkali-labile phosphate per mole of subunit. The phosphorylation sites have not been mapped, and the specificities of hepatic glycogen synthase kinases are not known. 

Carbohydrate SVI titrated as a monobasic acid, pK = 2.0, as expected for a phosphate diester, - and mild alkaline hydrolysis cleaved the labile, phosphate diester linkage, to produce a serologically inert monoester, [a]D -M06°, which was not precipitated by alcohol in the presence of calcium acetate. Acidic or enzymic hydrolysis of the monoester yielded a crystalline, nonreducing, chromatographically homogeneous tetrasaccharide which, after sequential methylation and acidic hydrolysis, yielded 2,3,4,6-... 

Reaction (a) is catalyzed by nucleoside phosphorylase. The reaction was originally detected by Kalckar as the phosphate-dependent splitting of nucleosides (I), and the detection of the resulting libose-l-phosphate depended upon the availability of the Lowry-Lopez method for determination of inorganic phosphate in the presence of labile phosphate esters. Although the enzjnne initially described from rat liver was not active with adenine, the corresponding beef enzyme was found to use... 

Citrullinase. The bacterial enzyme that degrades citrulline has been called citrullinase, citrulline ureidase and citrulline phosphorylase. The reaction requires inorganic phosphate, Mg++, and ADP, and ATP is formed together with NH3, CO2, and ornithine. Arsenate supports the breakdown of citrulline in the absence of phosphate and adenine nucleotides. The mechanism of the phosphorolysis was shown by Jones et to be straightforward the first products are ornithine and carbamyl phosphate, which had previously been considered to be too unstable to exist free. Carbamyl phosphate transfers its phosphate to ADP in a reversible kinase reaction, and the carbamyl group also can react with ornithine to form citrulline (VI). These reversible reactions explain the requirement for stoichiometric amounts of adenine nucleotides for a coupled reaction to remove the labile phosphate. Carbamic acid may equilibrate non-... 

Shortly after glucose-l-phosphate was discovered as the product of phosphorolysis of glycogen, Colowick and Cori observed an enzyme in extracts of muscle, fiver, brain, kidney, and yeast which converted the acid-labile phosphate of glucose-l-phosphate to an extremely acid-stable... 

As for the labile phosphate split off ATP, it was found that it contained a mixture of the terminal, highly active, and a second, less active fraction. On splitting off the terminal phosphate with an enzymic crayfish muscle suspension free of phosphate, Furchgott and Shorr obtained, after 30 minutes, a fraction having a specific activity of 100, while only one-half of the remaining labile phosphorus of ATP underwent renewal in the course of the experiment. 

Owing to the weak hydrophobicity of the PEO stationary phases and reversibility of the protein adsorption, some advantages of these columns could be expected for the isolation of labile and high-molecular weight biopolymers. Miller et al. [61] found that labile mitochondrial matrix enzymes — ornitine trans-carbomoylase and carbomoyl phosphate synthetase (M = 165 kDa) could be efficiently isolated by means of hydrophobic interaction chromatography from the crude extract. 

Phosphoramidate analogues of dideoxyribonucleoside phosphates (26) and trideoxyribonucleoside phosphates are acid labile and can be hydrolysed enzymically. Snake venom phosphodiesterase cleaves (26) to thymidine and 5 -deoxy-5 -aminothymidine (27 R = H). The latter presumably arises by spontaneous decomposition of the phosphoramidate (27 R = PO3H2) and P—O fission must have occurred during the initial hydrolysis. With acid or spleen phosphodiesterase, (26) gave Tp and (27 R = H), i.e. P—N fission occurred. 

Dihydroxybenzoate decarboxylase activity of these bacteria was induced specifically by 2,6-dihydroxybenzoate. The enzyme activity in a cell-free extract of A. tumefaciens 1AM 12048 was stable during storage at 4°C for 7 days in potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol. Different from 4-hydroxybenzoate decarboxylase and 3,4-dihydroxybenzoate decarboxylase, 2,6-dihydroxybenzoate decarboxylase was much less labile and barely... 

The isomerism existing between the pairs of nucleotides was attributed to the different locations of the phosphoryl residues in the carbohydrate part of the parent nucleoside,49 63 since, for instance, the isomeric adenylic acids are both hydrolyzed by acids to adenine, and by alkalis or kidney phosphatase to adenosine. Neither is identical with adenosine 5-phosphate since they are not deaminated by adenylic-acid deaminase,68 60 and are both more labile to acids than is muscle adenylic acid. An alternative explanation of the isomerism was put forward by Doherty.61 He was able, by a process of transglycosidation, to convert adenylic acids a" and 6 to benzyl D-riboside phosphates which were then hydrogenated to optically inactive ribitol phosphates. He concluded from this that both isomers are 3-phosphates and that the isomerism is due to different configurations at the anomeric position. This evidence is, however, open to the same criticism detailed above in connection with the work of Levene and coworkers. Further work has amply justified the original conclusion regarding the nature of the isomerism, since it has been found that, in all four cases, a and 6 isomers give rise to the same nucleoside on enzymic hydrolysis.62 62 63 It was therefore evident that the isomeric nucleotides are 2- and 3-phosphates, since they are demonstrably different from the known 5-phosphates. The decision as to which of the pair is the 2- and which the 3-phosphate proved to be a difficult one. The problem is complicated by the fact that the a and b" nucleotides are readily interconvertible.64,64... 

The position was somewhat clarified by the isolation of 2- and 3-O-phos-phonucleosides from ribonucleic acid hydrolyzates in 92 to 100% yields,134 and also by the demonstration that 5-O-phosphonucleosides are also present in enzymic digests.49, 197 Yet this information gave no indication of the nature of the alkali-labile linkages. Thus, while the majority of the experimental evidence pointed to the phosphoryl residues as being doubly esterified with adjacent nucleosides, two facts remained apparently inexplicable on the basis of this type of structure. First, ready fission by alkalis, and secondly, the absence of 5-phosphates from alkaline hydrolyzates and their presence in enzymic digests. Both these facts have been explained by Brown and Todd in the following way.92... 

A lot of biologic membrane systems and cellular organelles contain kinases, which transfer phosphate groups to proteins, especially to serine, threonine, and tyrosine residues. Self-phosphorylation of enzymes leading to acylphosphates or phosphoamides can be observed, too. With respect to their chemical stability, these phos-phoproteins are classified into acid-stable (alkali labile), hydroxyl-amine-sensitive, and acid labile. 

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