ABSTRACT
T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.
Subject(s)
Calorimetry , Polynucleotide 5'-Hydroxyl-Kinase , Kinetics , Calorimetry/methods , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , Polynucleotide 5'-Hydroxyl-Kinase/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Thermodynamics , Bacteriophage T4/enzymology , Diphosphates/chemistry , Diphosphates/metabolism , PhosphorylationABSTRACT
The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes a dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to regulate the PRC2 activity and hematopoiesis.
Subject(s)
DNA-Binding Proteins , Histones , Animals , Mice , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Hematopoiesis , Histones/metabolism , Methylation , Phosphorylation , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Beryllium ion elicits p53-mediated cell cycle arrest in some types of human cancer cells, and it is a potent inhibitor of GSK3 kinase activity. Paradoxically, Be2+ is regarded to have almost negligible aqueous solubility at physiological pH, due to precipitation as Be(OH)2. This study demonstrates that the interaction of Be2+ with serum proteins greatly increases its effective solubility. In typical serum-supplemented mammalian cell culture medium, Be2+ was soluble up to about 0.5 mM, which greatly exceeds the concentration needed for biological activity. Some biochemical studies require protein-free Be2+ solutions. In such cases, the inclusion of a specific inorganic counterion, sulfate, increased solubility considerably. The role of sulfate as a solubility-enhancing factor became evident during preparation of buffered solutions, as the apparent solubility of Be2+ depended on whether H2SO4 or a different strong acid was used for pH adjustment. The binding behavior of Be2+ observed via isothermal titration calorimetry was affected by the inclusion of sodium sulfate. The data reflect a "Diverse Ion Effect" consistent with ion pair formation between solvated Be2+ and sulfate. These insights into the solubility behavior of Be2+ at physiological and near-physiological pH will provide guidance to assist sample preparation for biochemical studies.