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1. A Primer on Protein Kinase Biochemistry 1.1. Phosphotransfer

Kinases or phosphotransferases are per definitionem enzymes that catalyze the transfer of the g-phosphate group from high-energy donor molecules such as adenosine-5′-triphosphate (ATP; sometimes guanosine-5′-triphosphate, GTP) preferentially to alcohol or hydroxy groups (OH) but also to nucleophilic centres of other functional groups of acceptor molecules, i.e. oxygen-, sulphur-, and nitrogen-containing ones (for details, see below; (1)). The process of attaching such a phosphorous-containing group is termed phosphorylation and leads to a phosphono or, in short, a

N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_2, © Springer Science+Business Media, LLC 2011

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N. Dissmeyer and A. Schnittger

phospho group in the acceptor molecule and is derived from phosphonic or phosphorous acid (H3PO3). This functional group is often also called a “phosphoryl” group which is not correct but regarded as acceptable (2). Together with the eight other families such as amino- and glycosyltransferases, kinases form the “transferase group” of enzymes classified as EC 2 according to the enzyme commission (EC). One of the largest groups of kinases are protein kinases (PKs), which act on and modify the activity of specific proteins. Depending on the species, PKs represent about 2–4% of all eukaryotic genes (Table 1 and references therein; (3, 4)) and it has been estimated that approximately one-third of all proteins in animal and yeast cells are phosphorylated on at least one position (5, 6). PKs, as phosphorous-transferring enzymes, constitute the group EC 2.7 which is further broken up with respect to the acceptor sites (Table  2): It is the hydroxy (OH), the carboxy(COOH), or nitrogenous (nitrogen-, N-containing) group of the side chains of the basic amino acids histidine (His; H), arginine (Arg; R), and lysine (Lys; K) as well as the sulphur-containing group of cysteine (Cys; C) side chains that serve as phosphoacceptors typically located on the surface of specific target molecules or substrates. In His, it is the imidazole (C3H3N2+) sidechain, in Arg, a guanidinium (CH6N3+) group, in Lys, the terminal amino (NH2) group, and in Cys, the sulfhydryl or hydrosulphide group (SH). Besides phosphorylation on the OH-groups of Ser/Thr and Tyr that forms a phosphate ester bond (O-phosphono group or O-phosphate; Fig. 1a, b), phosphorylation of the nitrogen of His, Arg, and Lys forming a phosphoramidate linkage (N-phosphono group or N-phosphate; Fig.  1c), the sulphur of Cys forming a phosphate thioester (S-phosphono group, thio or S-phosphate), and the carboxy group of aspartate (Asp; D) and glutamate (Glu; E) forming an acid anhydride (acyl phosphate) can also occur. In eukaryotic cells, hydroxy-linked phosphorylation is by far the most studied and thio and acyl phosphates are extremely rare (7). Analysis of the abundance of phosphorylation sites revealed that the most prominen