Size and charge requirements for kinetic modulation and actin binding by alkali 1-type myosin essential light chains

The alkali 1-type isoforms of myosin essential light chains from vertebrate striated muscles have an additional 40 or so amino acids at their N terminus compared with the alkali 2-type. Consequently two light chain isoenzymes of myosin subfragment-1 can be isolated. Using synthesized peptide mimics of the N-terminal region of alkali 1-type essential light chains, we have found by ¹H NMR that the major actin binding region occurred in the N- terminal four residues, APKK.... These results were confirmed by mutating this region of the human atrial essential light chain, resulting in altered actin-activated MgATPase kinetics when the recombinant light chains were hybridized into rabbit skeletal subfragment 1. Substitution of either Lys³ or Lys⁴ with Ala resulted in increased K(m) and k(cat) and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys⁴ with Asp reduced actin binding and increased K(m) and k(cat) still further. Alteration of Ala¹ to Val did not alter the kinetic parameters of the hybrid subfragment 1 or the essential light chain's ability to bind actin. Furthermore, we found a significant correlation between the apparent K(m) for actin and the k(cat) for MgATP turnover for each mutant hybrid, strengthening our belief that the binding of actin by alkali 1-type essential light chains results directly in modulation of the myosin motor.