SUBSTRATE-SPECIFICITY OF DEUBIQUITINATING ENZYMES - UBIQUITIN C-TERMINAL HYDROLASES

Ubiquitin C-terminal hydrolases (UGH) are deubiquitinating enzymes whi ch hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cle aved slowly. Ubiquitinyllysine derivatives linked by the alpha- or eps ilon-amino group are hydrolyzed at identical rates. Isozyme-specific h ydrolytic preferences are only evident when the leaving group is large . The ubiquitin gene products can be cotranslationally processed by on e or both of these UCH isozymes, and purified UbCEP52 can be hydrolyze d by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because o f the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydr olyze large ubiquitin derivatives such as N-epsilon-ubiquitinyl-cytoch rome-c, N-epsilon-(K48)polyubiquitinyl-lysozyme, or an N-alpha-ubiquit inyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly an d preferentially cleave small leaving groups such as amino acids and o ligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological rol e of UCH is to hydrolyze small adducts of ubiquitin and to generate fr ee monomeric ubiquitin from ubiquitin proproteins, but not to deubiqui tinate ubiquitin-protein conjugates or disassemble polyubiquitin chain s.