The results of this study show that premenopausal women with endometrial carcinoma may be treated successfully with progestin therapy alone as primary therapy to preserve childbearing potential.

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.The p53 gene product is an important tumor suppressor and is inactivated by deletion or mutation in approximately 50% of all human cancer. The p53 protein functions as a transcription factor that binds DNA and induces the expression of a number of genes involved in cell growth arrest, DNA repair, and apoptosis. p53 is maintained at low steady-state levels in the cell and is induced and activated post-translationally by various signaling pathways that respond to cellular stress (1, 2). Cellular insults that initiate the stress response and activate p53 include DNA-damaging agents (chemical, UV, and ionizing radiation), oxygen stress, or the inappropriate activation of oncogenes. Activated p53 induces either growth arrest or apoptosis depending on the extent and duration of signals generated from the damage (3, 4). The post-translational modifications involved in p53 activation and increased steady-state levels of the protein include phosphorylation, dephosphorylation, acetylation, sumoylation, and ubiquitination. The stability and half-life of p53 are tightly regulated by mdm2 and the ubiquitin-proteasome pathway (5-7). Recent evidence suggests that mdm2 is an E3 1 ubiquitin ligase for p53 (8, 9). A number of critical regulatory proteins in the cell are modified by ubiquitin (Ub) conjugation. Proteasomal degradation of key regulatory proteins control biological events involving the cell cycle, differentiation, immune responses, DNA repair, chromatin structure, and apoptosis (10). The initial step in the Ub cascade is the activation of Ub by the ubiquitin-activating enzyme (E1). E1 hydrolyzes ATP to AMP and pyrophosphate to generate a thioester bond between the active site Cys of E1 and the carboxylterminal Gly of Ub. The activated Ub is transf...

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.

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