Characterization of the Novel Catalytic and Regulatory Properties of the Alpha‐Kinase Domain of Dictyostelium Myosin Heavy Chain Kinase A

Dictyostelium myosin heavy chain kinase A (MHCK‐A) is a member of the alpha‐kinase family of atypical Ser/Thr protein kinases. The X‐ray crystal structure of the alpha‐kinase domain of MHCK A (A‐CAT: residues 552–841) shows that an aspartylphosphate residue is present within the active site, indicating that the catalytic mechanism may proceed through the formation of a phosphoenzyme intermediate. Further structural analysis reveals that A‐CAT can hydrolyze ATP to adenosine. Regulation of A‐CAT is predicted to involve the binding of a Mg2+ ion to a loop, termed, the N/D‐loop, that regulates access to the active site cleft. In addition, A‐CAT contains a positively‐charged phosphate‐binding pocket that functions to potently activate kinase and ATPase activity. A critical autophosphorylation site (T825) located in a disordered sequence C‐terminal to the alpha‐kinase domain is predicted to activate A‐CAT by providing a ligand for the phosphate‐binding pocket. A fluorescent probe has been used to analyze nucleotide binding to the A‐CAT active site. In addition, a fluorescent probe has been linked to the N/D‐loop in order to directly measure the ability of the loop to undergo a divalent cation‐induced conformational change. The effects of altering the position of the phosphothreonine within the C‐terminal tail on A‐CAT activity is also examined.