Phosphorylation, biotin

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

The key enzyme in fatty acid synthesis is acetyl CoA carboxylase (see p. 162), which precedes the synthase and supplies the malonyl-CoA required for elongation. Like all carboxylases, the enzyme contains covalently bound biotin as a prosthetic group and is hormone-dependently inactivated by phosphorylation or activated by dephosphorylation (see p. 120). The precursor citrate (see p. 138) is an allosteric activator, while palmitoyl-CoA inhibits the end product of the synthesis pathway. 

The conversion of pyruvate to acetyl CoA and C02 A. is reversible. B. involves the participation of lipoic acid. C. is activated when pyruvate dehydrogenase complex is phosphorylated by a protein kinase in the pres ence of ATP. D. occurs in the cytosol. E. depends on the coenzyme biotin. Correct answer = B. Lipoic acid is an intermedi ate acceptor of the acetyl group formed in the reaction. Pyruvate dehydrogenase complex cat alyzes an irreversible reaction that is inhibited when the enzyme is phosphorylated. The enzyme is located in the mitochondrial matrix. 

Have we checked all of the possibilities for the mechanism of biotin carboxylation Kruger and associates62 63 suggested that biotin, as a ureido anion, might add to bicarbonate to form a highly unstable intermediate which, however, could be phosphorylated by ATP (Eq. 14-10, steps a and b). This intermediate could undergo elimination of inorganic phosphate... 

Kinase or phosphatase assays based on the AlphaScreen principle are similar to TR-FRET assays in that they usually require a biotinylated substrate peptide and an anti-phosphoserine or tyrosine antibody. These two reagents are sandwiched between biotin and protein A-functionalized acceptor and donor beads. A kinase assay would show an enzyme-dependent increase in antibody binding (and thus signal) over time and a phosphatase assay would show an enzyme-dependent decrease in antibody binding over time. In some cases, the phosphorylation of an epitope will block the antibody binding and thus a phosphatase assay in principle can be constructed as a signal increase assay (Von Leoprichting and Kumpf, 2004 Warner et al., 2004). 

The thiazole ring is synthesized from a pentulose or deoxypentulose 5-phosphate and either glycine or tryptophan, depending on the organism. Incorporation of sulfur leads to formation of hydroxymethyl thiazole, which is then phosphorylated. The sulfur comes from cysteine and is incorporated by formation of a thiocarboxylate at the carboxyl terminal of the enzyme, unlike biotin synthesis (Section 11.1.1), where an iron-sulfur cluster at the active site of the enzyme is the donor. 

The role of ATP in the carboxylation of biotin is unclear. It is possible that biotin is O-phosphorylated during the carboxylation reaction. However, evidence suggests that the immediate reactive species that carboxylates biotin is carboxyphosphate, as in the (biotin-independent) reaction of carbamyl phosphate synthetase in urea and pyrimidine synthesis. 

Figure 4.46 shows the first step (acetyl-CoA carboxylase catalyzed) in the fatty acid synthesis pathway. The enzyme is biotin-requiring, and the product is malonyl-CoA. Note that the activities of about 100 different enzymes have been foxmd to be controlled by phosphorylation (Shacter et ah, 1986). In all cases, the phosphorylation is reversible. The phosphate donor may be ATP or GTP. 

Another possibility within this class of mechanism involves the initial reaction of biotin with ATP, forming ADP and a phosphorylated biotin species. It has been proposed, based on model studies, that such a species would be O-phosphobiotin (52,53). This reacts with bicarbonate to produce iV-carboxybiotin and inorganic phosphate (Scheme 20). The transfer of oxygen from bicarbonate occurs in the second step in this case. Models for the 0-phosphorylation of biotin suggest that such a process can occur readily. 

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