EXPRESSION, PURIFICATION FROM INCLUSION-BODIES, AND CRYSTAL CHARACTERIZATION OF A TRANSITION-STATE ANALOG COMPLEX OF ARGININE KINASE - A MODEL FOR STUDYING PHOSPHAGEN KINASES

Phosphagen kinases catalyze the reversible transfer of a phosphoryl gr oup between guanidino phosphate compounds and ADP, thereby regeneratin g ATP during bursts of cellular activity. Large quantities of highly p ure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in art hropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size excl usion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a tr ansition state analog complex. Near complete native diffraction data h ave been collected to 1.86 Angstrom resolution. Substitution of a reco mbinant source for a natural one, improvement in the purification, and data collection at cryo temperatures have all yielded significant imp rovements in diffraction.