Protein Structure and Function Lecture 8 Notes: Phosphorylation Phosphorylation - Levine (1906) demonstrated that the protein vitellin (a major yolk protein) contained a phosphate group. Levine and Lipman (1933) obtained phosphoserine from casein via hydrolysis and then Friedkin and Lehninger (1949) discovered that rat liver particles incorporate radioactive phosphate into phosphoprotein. Similarly, Burnett and Kennedy (1954) discovered rat liver mitochondria also incorporate radioactivity from 32P, presumed via AT32P into casein serine and that casein was more receptive than the other 5 proteins studied. Krebs and Fischer (1955) discovered that the activity of glycogen phosphorylase is regulated by reversible phosphorylation. Finally, the role of JAK (Janus) kinases in JAK-STAT regulatory systems (i.e. the role of phosphorylation in gene expression) was elucidated in the 1990s. - 30-65% of proteins in the human genome and around 50% of proteins in the yeast genome may be phosphorylated. Possibly up to 75% of all PTMs are phosphorylations (followed by N- glycosylation). Therefore, this is an extremely important PTM. - The discovery of the regulation of glycogen phosphorylase by Krebs and Fischer (1938-56) OH alt+4) bond OH outlined the process by which glycogen is HO HO OH glycogen OH OH phosphorylase HO C HO HO converted to a-D-glucose-1-phosphate and a OH OH glycogen n-mer. The substrate is glycogen, a O Jn OH linear partially branched polymer of glucose glycogen (n+1)-mer @-o-glucose-1-phosphate glycogen n-mer units and the main links are a1-4 links between glucose units. Glycogen phosphorylase splits the terminal sugar from the glycogen polymer chain, producing a-D-glucose-1-phosphate (i.e. degrades glycogen). - They also discovered (1956) that this enzyme exists in two forms (a and b) which differ in activityff a is more active than b. It was demonstrated that Phosphorylase b Active these forms can be interconverted, and that the reaction is site dependent on the presence of a metal2+ group and ATP. In 2 ATP 2 ADP R state 1. 1959, they demonstrated that the interconversion is carried out by two enzymes, one which incorporates a phosphate group from ATP and the other which removes this (a-P2 = b). The two forms thus differ in the presence of a phosphate group on the enzyme. The enzymes which remove P from proteins (phosphatases) have the enzyme classification 2 ATP 2 ADP E.C.3.1.3.X whereas enzymes which incorporate phosphates T state P into proteins (kinases) have the classification E.C.2.7.1.X. Phosphorylase a Phosphorylase a and b can exist themselves in two different states (relaxed and tense). The enzymes are regulated not only by phosphorylation but also by interaction with substrates and products. Phosphorylase a (active form) is phosphorylated on serine 15 of each of the two subunits. This modification causes a conformational change, especially at the N-terminal which promotes a more active tetramer from the dimer and abolishes AMP (+) and ATP (-) regulation. This form favours the more active R (relaxed) state. Phosphorylase b (less active)
is not phosphorylated and exists predominantly in the T (tense) state. Phosphorylase b is allosterically activated by AMP and is inhibited by