Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization

The mdm2 gene product is an important regulator of p53 function and stabili ty. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of md m2 is critical for ligase activity. Ubiquitin (Uh) conjugation is a general targeting modification and poly-ubiquitin chains specifically target prote ins to the proteasome for degradation. In this report, we show that the mul tistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed wi th individual components and the p53-Ub((n)) is qualitatively similar to p5 3-Ub((n)), detected in lactacystin-treated cells. Surprisingly, we find tha t p53 is modified with multiple mono-ubiquitin moieties as opposed to a pol y-ubiquitin chain. Finally, kinetic analysis indicates the transfer reactio n proceeds either through a modified Ping Pong mechanism involving requisit e enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi m echanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric chang es in enzyme structure.