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 stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep 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 with individual components and the p53-Ub((n)) is qualitatively similar to p53-Ub((n)) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.
Lai
Z
Z
Cancer Research, DuPont Pharmaceuticals Company, Glenolden, Pennsylvania 19036, USA.
Ferry
K V
KV
Diamond
M A
MA
Wee
K E
KE
Kim
Y B
YB
Ma
J
J
Yang
T
T
Benfield
P A
PA
Copeland
R A
RA
Auger
K R
KR
D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000502:physiology*; D051736:Proto-Oncogene Proteins c-mdm2; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*
D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D005033:Ethylmaleimide