A necessary step in ubiquitin-dependent proteolysis is the addition of a polyubiquitin chain to the target protein. This ubiquitinated protein is degraded by a multisubunit complex known as the 26S proteasome. The polyubiquitin chain is probably not released until a late stage in the proteolysis by the proteasome. It is subsequently disassembled to yield functional ubiquitin monomers. Here we present evidence that a 93 kDa protein, isopeptidase T, has the properties expected for the enzyme which disassembles these branched polyubiquitin chains. Protein and cDNA sequencing revealed that isopeptidase T is a member of the ubiquitin specific protease family (UBP). Isopeptidase T disassembles branched polyubiquitin chains (linked by the G76-K48 isopeptide bond) by a sequential exo mechanism, starting at the proximal end of the chain (the proximal ubiquitin contains a free carboxyl-terminus). Isopeptidase T prefers to disassemble chains in which there is an intact and unblocked RGG sequence at the C-terminus of the proximal subunit. Rates of disassembly are reduced when G76 of the proximal ubiquitin is modified, for example, by ligation to substrate protein, by esterification, by replacement of the proximal glycine with alanine (G76A), or by truncation. Linear proubiquitin is only a poor substrate. Observed rates and specificity are consistent with isopeptidase T playing a major role in disassembly of polyubiquitin chains. The high discrimination against chains that are blocked or modified at the proximal end indicates that the enzyme acts after release of the chains from conjugated proteins or degradation intermediates. Thus, the proteolytic degradation signal is not disassembled by isopeptidase T before the ubiquitinated protein is degraded. These (and earlier) results suggest that UBP isozymes may exhibit significant substrate specificity, consistent with a role in the regulated catabolism of the polymeric ubiquitin, including the polyubiquitin protein degradation signal.

A nonhydrolyzable analogue of ubiquitin adenylate has been synthesized for use as a specific inhibitor of the ubiquitination of proteins. Ubiquitin adenylate is a tightly bound intermediate formed by the ubiquitin activating enzyme. The inhibitor adenosyl-phospho-ubiquitinol (APU) is the phosphodiester of adenosine and the C-terminal alcohol derived from ubiquitin. APU is isosteric with the normal reaction intermediate, the mixed anhydride of ubiquitin and AMP, but results from the replacement of the carbonyl oxygen of Gly76 with a methylene group. This stable analogue would be expected to bind to both ubiquitin and adenosine subsites and result in a tightly bound competitive inhibitor of ubiquitin activation. APU inhibits the ATP-PPi exchange reaction catalyzed by the purified ubiquitin activating enzyme in a manner competitive with ATP (Ki = 50 nM) and noncompetitive with ubiquitin (Ki = 35 nM). AMP has no effect on the inhibition, confirming that the inhibitor binds to the free form of the enzyme and not the thiol ester form. This inhibition constant is 10-fold lower than the dissociation constants for each substrate and 30-1000-fold lower than the respective Km values for ubiquitin and ATP. APU also effectively inhibits conjugation of ubiquitin to endogenous proteins catalyzed by reticulocyte fraction II with an apparent Ki of 0.75 microM. This weaker inhibition is consistent with the fact that activation of ubiquitin is not rate limiting in the conjugation reactions catalyzed by fraction II. APU is similarly effective as an inhibitor of the ubiquitin-dependent proteolysis of beta-lactoglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)