ACTIVATION OF MYOSIN LIGHT-CHAIN KINASE AND NITRIC-OXIDE SYNTHASE ACTIVITIES BY ENGINEERED CALMODULINS WITH DUPLICATED OR EXCHANGED EF HANDPAIRS

We have constructed three engineered calmodulins (CaMs) in which the t wo EF hand pairs have been substituted for one another or exchanged: C aMNN, the C-terminal EF hand pair (residues 82-148) has been replaced by a duplication of the N-terminal. pair (residues 9-75); CaMCC, the N -terminal pair has been replaced by a duplication of the C-terminal pa ir; CaMCN, the two EF had pairs have been exchanged, Skeletal muscle m yosin light chain kinase (skMLCK) activity is activated to 75% of the maximum level by CaMCC and to 45% of the maximum level by CaMCN and is not significantly activated by CaMNN; K-act or for values for the eng ineered CaMs are 2-3.5 nM. Smooth muscle myosin light chain kinase act ivity (gMLCK) is fully activated by CaMCN and is not significantly act ivated by either CaMNN or CaMCC; the K-act value for CaMCN is 2 nM and the K-i values for CaMNN and CaMCC are 10 and 40 nM, respectively, Ce rebellar nitric oxide synthase activity (nNOS) is fully activated by C aMNN and CaMCN and is not significantly activated by CaMCC; the engine ered CaMs have K-act or K-i values for this enzyme activity of 2-8 nM. These results indicate that the EF hand pairs contain distinct but ov erlapping sets of determinants for binding and activation of enzymes, with the greater degree of overlap in determinants for binding. Furthe rmore, while the structural changes associated with swapping the EF ha nd pairs do not affect activation of nNOS or gMLCK activities, they si gnificantly reduce activation of skMLCK activity, indicating that this process requires specific determinants in CaM outside the EF hand pai rs.