ALTERED METHYLATION SUBSTRATE KINETICS AND CALCIUM-BINDING OF A CALMODULIN WITH A VAL136-]THR SUBSTITUTION

Calmodulin is trimethylated on Lys115 by a specific calmodulin methylt ransferase. Previously, it was shown that the can 12 mutant (Ile136--> Thr) of Paramecium has a decreased level of methylated Lys115 [Lukas, T. J., Friedman, M. W., Kung, C. & Watterson, D. M. (1989) Proc. Natl Acad. Sci. USA Sd, 7331-7335]. To investigate how this substitution af fects calmodulin structure, function and recognition by the calmodulin methyltransferase, a calmodulin with a Thr136 substitution ([Thr136]c almodulin) was expressed in Escherichia coli in an unmethylated form f or in vitro enzyme activator, calcium binding and methylation kinetic analyses. [Thr136]calmodulin was indistinguishable from wild-type calm odulin in saturating (1 mM) calcium in its ability to activate calmodu lin-dependent enzymes and in its steady-state kinetic properties with isolated calmodulin methyltransferase. However, [Thr136]calmodulin did show two defects: a complete inability to be methylated in the absenc e of calcium; and defective calcium binding. As a result, an approxima te 10-fold shift in the K-0.5 values for calcium dependence of enzyme activation (shifted from 1.1 mu M to 9.1 mu M of Ca2+ for NAD kinase) and methylation (from 0.71 mu M to 7.2 mu M of Ca2+ in 0.15 M K+, 2 mM Mg2+) were observed. Non-denaturing electrophoresis and Tyr138 spectr oscopic measurements suggest a difference in the conformation of the c alcium-depleted structures of normal calmodulin and [Thr136]calmodulin . Overall, the results suggest that the mutation in this conserved pos ition in the COOH-terminal hydrophobic core lowers calcium-binding aff inity and alters the calcium-depleted structure leading to decreased m ethylation at physiological Ca2+ concentrations.