Ubiquitin thioester

After the formation of an acyl adenylate, the similarities between MoeB and El appear to come to an end (Figure 3.2B). In the El enzymes an active-site cysteine residue attacks the ubiquitin adenylate forming the El-ubiquitin thioester. E. coli MoeB contains nine cysteine residues, four of which are involved in coordinating the zinc atom. Sequence alignments show that among the remaining cysteines... 

One of the central mechanistic questions regarding ubiquitination has been whether the reaction utilizes general acid/base catalysis, possibly in a manner analogous to the catalysis of peptide-bond cleavage. For example, an acidic catalytic residue could deprotonate the substrate lysine and make it a better nucleophile for attacking the ubiquitin thioester bond. In addition, a basic catalytic residue could polarize the thioester bond making the carbonyl carbon a better electrophile, and... 

ScHEFFNER, M., NuBER, U. and Huibregtse, j. M. Protein ubiquitination involving an El—E2—E3 enzyme ubiquitin thioester cascade. Nature 1995, 373, 81-3. 

Natural products have recently been shown to interfere with specific steps in both the ubiquitination and de-ubiquitination of proteins, effectively preventing their recognition and degradation by the 26S proteasome. Panepophenanthrin (20), isolated from the mushroom Panus radis and himeic acid A (21), isolated from a marine strain of Aspergillus sp., have been shown to interfere with the ubiquitin-activating function of El enzymes. In this ATP-driven process, an adenylated ubiquitin-AMP intermediate is first formed. This ubiquitin intermediate then forms an activated ubiquitin-Cys thioester in the active site of El. Both compounds have been shown to interfere with the formation of the adenylated ubiquitin-AMP intermediate that is required to form the activated ubiquitin thioester. 

Figure29-1. Partial reactions in the attachment of ubiquitin (UB) to proteins. (1) The terminal COOH of ubiquitin forms a thioester bond with an -SH of E, in a reaction driven by conversion of ATP to AMP and PP. Subsequent hydrolysis of PP by pyrophosphatase ensures that reaction 1 will proceed readily. (2) A thioester exchange reaction transfers activated ubiquitin to Ej. (3) E3 catalyzes transfer of ubiquitin to e-amino groups of lysyl residues of target proteins.

Fig. 1. A schematic diagram outlining the hierarchic structure of the ubiquitin system. In an ATP-dependent manner a thioester bond is formed between the C-terminus of ubiquitin and an internal cystein residue of the ubiquitin-activating enzyme. Subsequently, ubiquitin is transferred to a member of the family of ubiquitin-conjugating enzymes, which are also able to form a thioester bond with ubiquitin. The third class of enzymes, the ubiquitin ligases, direct ubiquitin to the proteolytic substrates. Different families of this class of enzymes are known, some of which are also able to form a thioester intermediate with ubiquitin (HECT-domain ligases). The final ubiquitin-substrate linkage is an isopeptide bond between the C-terminus of ubiquitin and internal lysine residues in the substrate proteins...

Fig. 2.15A. Pattern of ubiquitinylation of proteins and degradation in the proteosome. Ubiquitin (Ub)is initially activated by an enzyme El, whereby the C-terminal carboxyl group of ubiquitin becomes attached to a SH group of El via a thioester bond. Ubiquitin is then transferred from El-Ub to E2, from which it is transferred with the help of E3 to the target protein. Several ubiquitin molecules can attach to the target protein in a hnear or in a crosshnked fashion. The mono- or polyubiquitinylated protein is degraded to peptides in the 26S proteosome. In the above diagram the filled circles represent the ubiquitin residues attached to the target protein. K lysine residues of the target protein.

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