2520 - Protein Structure and Function Lecture 9 Protein Phosphorylation Discovery of phosphorylation: 1906 Phoebus Levine showed that a protein vitelline (major egg yolk protein) contained a phosphate group 1933 Levine & Felix Lipman obtained phosphoserine from casein by hydrolysis 1949 Friedkin, M., and Lehninger, A. L., rat liver particles incorporate radioactive phosphate into phosphoprotein 1954 G Burnett & E P Kennedy, rat liver mitochondria incorporate radioactivity from 32P phosphate, presumed via AT32P, into casein serine; casein more receptive than 5 other proteins studied (why?) 1955 E Krebs and E H Fischer, the activity of glycogen phosphorylase is regulated by reversible phosphorylation, Nobel Prize in Physiology or Medicine 1992 ~1990 JAK (Janus) kinases, JAK-STAT regulatory system elucidated 30-65% of proteins in the human genome and ~50% of proteins in the yeast genome may be phosphorylated; possibly 75% of PTMs are phosphorylations Glycogen phosphorylase reaction: - E Krebs and E Fischer - Nobel prize in 1992. - It is an enzyme that chops up glycogen to produce glucose-1-phosphate. - It became clear the GP exists in two forms - one active and another inactive. - The difference in their activity was difficult to assess - it wasn't known that phosphorylation could be reversed by phosphatases - it was a dynamic system. Phosphorylation - effective means of regulating activities of target proteins. - What makes protein phosphorylation so valuable in regulating protein function? - The free energy of phosphorylation is large - phosphorylation can change the conformational equilibrium between different functional states by a large factor (104) - the energy expenditure allows for a stark shift from one conformation to another. - A phosphoryl group adds 2 negative charges to a modified protein - these new charges may disrupt electrostatic interactions in the unmodified protein and allow new electrostatic interactions to be formed - this greatly alters substrate binding and catalytic activity. - A phosphoryl group can form 3 or more hydrogen bonds - the tetrahedral geometry of a phosphoryl group makes these bonds highly directional, allowing for specific interactions with hydrogen bond donors. - Phosphorylation and dephosphorylation can take place in less than one second or over an hour - kinetics can be changed to meet the timing needs of a process. - Phosphorylation often produces highly amplified effects - a single activated kinase can phosphorylate hundreds of target proteins. - ATP is the cellular energy currency.
The Chemistry of Protein Phosphorylation - This is called the kinase reaction. - The activity (a)ffiinactivity (b) of glycogen phosphorylase (i.e. the two forms) depended on whether or not the protein itself was phosphorylated. - Requires a kinase to attach a phosphate group to the GP and a phosphatase to reverse this. - Attachment of a phosphate group to a protein: o ATP (phosphate donor) and a recipient side chain on a protein - eg. serine - hydroxyl group is the recipient. o The phosphate terminus of ATP is transferred to the serine to give a phosphorylated protein = phosphoserine - and ADP. We