Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.     

Acidic residues comprise part of the myosin light chain-binding site on skeletal muscle myosin light chain kinase

Myosin light chain kinase is a Ca2+/calmodulin-dependent protein kinase which exhibits a very high degree of protein substrate specificity. The regulatory light chain of myosin is the only known physiological substrate of the enzyme. Based upon epitope mapping of monoclonal antibodies which inhibit kinase activity competitively with respect to the light chain substrate, residues 235-319 of the rabbit skeletal muscle kinase have been proposed to contain a light chain-binding site (Herring, B. P., Stull, J. T., and Gallagher, P. J. (1990) J. Biol. Chem. 265, 1724-1730). With the expression of a truncated kinase, we have further localized this putative binding site to residues 235-294. Mutation of acidic residues at positions 269 and 270 of the kinase resulted in a 10-fold increase in the Km value for the myosin light chain, with no significant change in the Vmax value. In contrast, altering a cluster of acidic amino acids at positions 261-263 had little effect on the Km value for the myosin light chain. These results suggest that residues 269 and 270 may be involved in protein-substrate binding. Interestingly, these residues, located amino-terminal of the homologous catalytic core (positions 302-539), are in a region which is highly conserved among myosin light chain kinases, but not other protein kinases. It is probable that the homologous catalytic core contains structural elements required for phosphotransferase activity. The catalytic domain of myosin light chain kinase would therefore include these conserved elements together with additional specific substrate-binding residues.     

Domain characterization of rabbit skeletal muscle myosin light chain kinase.

Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal "tail," of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235-319 and 165-173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Characterization of chicken skeletal muscle myosin light chain kinase. Evidence for muscle-specific isozymes

Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.     

Molecular characterization of a mammalian smooth muscle myosin light chain kinase.

A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.     

Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase.

It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.     

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.     

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.     

Homologous calcium-binding proteins in the activation of skeletal, cardiac, and smooth muscle myosin light chain kinases

In order to identify the physiological regulator of calcium dependent myosin light chain kinases of cardiac, skeletal, and smooth muscles, the effects of the three homologous calciproteins, calmodulin, troponin C, and parvalbumin, on the kinases isolated from bovine myocardium, rabbit skeletal muscle, and turkey gizzard were examined. Only calmodulin was effective in stimulating the cardiac, skeletal, or smooth muscle kinase; troponin C and parvalbumin exhibited no activation of any of the three kinases, even when examined at concentrations as high as 10-(5) M. It is concluded that calmodulin is the specific regulator of myosin light chain kinase in cardiac, skeletal, and smooth muscle.     

Purification and characterization of bovine cardiac calmodulin-dependent myosin light chain kinase.

Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately 1200-fold with 16% yield by a three-step procedure. The approximate content of soluble myosin light chain kinase in heart is calculated to be 0.63 microM. The isolated kinase is active only as a ternary complex consisting of the kinase, calmodulin, and Ca2+; the apparent Kd for calmodulin is 1.3 nM. The enzyme also exhibits a requirement for Mg2+ ions. Myosin light chain kinase is a monomeric enzyme with Mr = 85,000. The enzyme exhibits a Km for ATP of 175 microM, and a K0.5 for the regulatory light chain of cardiac myosin of 21 microM. The optimum pH is 8.1. Kinase activity is specific for the regulatory light chain of myosin. The specific activity of the isolated enzyme (30 nmol 32P/min/mg of protein) is considerably less than and corresponding values reported for the skeletal and smooth muscle light chain kinases. This is probably due to proteolysis during extraction of the myocardium, a phenomenon which has, as yet, proven impossible to eliminate. In contrast to the smooth muscle enzyme (Adelstein, R.S., Conti, M.A., Hathaway, D.R., and Klee, C.B. (1978) J. Biol. Chem. 253, 8347-8350), the cardiac kinase is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Properties of myosin light chain kinase prepared from rabbit skeletal muscle by an improved method

Myosin light chain kinase was prepared from rabbit skeletal muscle. DEAE-Sephadex, calmodulin-Sepharose 4B affinity gel and Ultrogel AcA 34 were used for the purification. It took 3 days for the preparation, and 6.2 mg of myosin light chain kinase was isolated from 600 g of frozen muscle. The molecular weight of the myosin light chain kinase estimated by sedimentation equilibrium analysis was 103,000 +/- 4,100. The isoelectric point was 5.0. Chemical modification of cysteine residues did not affect the catalytic activity, but modification of tyrosine residues diminished the activity. In order to activate myosin light chain kinase, it was necessary to bind calmodulin in an equimolar ratio and the dissociation constant was estimated to be 3.6 nM. The optimum pH for the catalytic activity was 7.5, and the activity was inhibited by NaCl and KCl. In the presence of 2.74 mg/ml myosin light chain and 75 mM KCl, the catalytic activity was found to be 88 s-1. The Vm and Km at 0.14 M KCl were 100 s-1 and 53 microM, respectively, for the isolated light chain as substrate and 70-80 s-1 and 19 microM for myosin as substrate.     

A comparative study of the myosin light chain kinases from myoblast and muscle sources. Studies on the kinases from proliferative rat myoblasts in culture, rat thigh muscle, and rabbit skeletal muscle.

Myosin light chain kinases have been isolated from rat thigh and rabbit skeletal muscle and cultured rat myoblasts. From these preparations, two types of kinases can be distinguished: calcium-dependent and calcium-independent. Both types of kinases can phosphorylate isolated P-light chains of myosin from several sources (skeletal muscle, cardiac muscle, and platelet). Data are shown which support the phosphorylation of the same site on the non-muscle P-light chains by both types of kinases. The rates of these reactins are, however, different for the two types of kinases. Kinetic analysis of the myoblast kinase shows differing affinities for various P-light chains (non-muscle greater than cardiac greater than skeletal). In the proliferative rat myoblast, phosphorylation of myosin is a prerequisite for actin activation of the myosin ATPase activity.     

Functional assembly of fragments from bisected smooth muscle myosin light chain kinase

The C-terminal regulatory segment of smooth muscle myosin light chain kinase folds back on its catalytic core to inhibit kinase activity. This regulatory segment consists of autoinhibitory residues linking the catalytic core to the calmodulin-binding sequence and perhaps additional C-terminal residues including an immunoglobulin-like motif. However, mutational and biochemical analyses showed no specific involvement of residues C-terminal to the calmodulin-binding sequence. To obtain additional insights on the proposed mechanisms for autoinhibition and Ca(2+)/calmodulin activation of the kinase, the polypeptide backbone chain of myosin light chain kinase was cleaved by genetic means to produce N- and C-terminal protein fragments. The N-terminal fragment containing the catalytic core was catalytically inactive when expressed alone. Co-expression of the N-terminal fragment with the C-terminal fragment containing the regulatory segment restored kinase activity. Deletion of the autoinhibitory linker residues without or with the calmodulin-binding sequence prevented restoration of kinase activity. In the presence or absence of Ca(2+)/calmodulin, regulatory segment binding occurred through the linker region connecting the catalytic core to the calmodulin-binding sequence. Collectively, these results indicate that residues C-terminal to the calmodulin-binding sequence (including the immunoglobulin-like motif) are not functional components of the regulatory segment. Furthermore, the principal autoinhibitory motif is contained in the sequence linking the catalytic core of myosin light chain kinase to the calmodulin-binding sequence.     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Structural studies of rabbit skeletal muscle myosin light chain kinase with monoclonal antibodies

Monoclonal antibodies directed against rabbit skeletal muscle myosin light chain kinase have been used to study the domains of this kinase. Specificity of nine monoclonal antibodies against rabbit skeletal muscle myosin light chain kinase was demonstrated by immunoblot analysis and immunoadsorption of kinase activity. None of the antibodies reacted by immunoblot analysis with either chicken skeletal or rabbit smooth muscle myosin light chain kinases. Epitope mapping of trypsin-digested rabbit skeletal muscle myosin light chain kinase showed that antibodies 2a, 9a, 9b, 12a, 12b, 16a, and 16b are directed against the 40-kDa catalytic domain. In addition, these seven antibodies reacted with sites that are clustered within a 14-kDa fragment of the kinase generated by Staphylococcus aureus V8 protease digestion. Two monoclonal antibodies, 14a and 19a, reacted with two distinct epitopes located within the inactive, asymmetric trypsin fragment. Six of nine monoclonal antibodies (2a, 9a, 9b, 12a, 12b, and 14a) inhibited kinase activity. Kinetic analyses demonstrated that antibodies 2a, 12a, and 14a inhibited kinase activity competitively with respect to myosin phosphorylatable light chain; 2a, 12a, and 14a exhibit noncompetitive inhibition with respect to calmodulin. These data suggest that monoclonal antibodies 2a, 12a, and 14a bind at or adjacent to the active site of the kinase.     

Identification, phosphorylation, and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin

At relatively high concentrations of myosin light chain kinase, a second site on the 20,000-dalton light chain of smooth muscle myosin is phosphorylated (Ikebe, M., and Hartshorne, D. J. (1985) J. Biol. Chem. 260, 10027-10031). In this communication the site is identified and kinetics associated with its phosphorylation and dephosphorylation are described. The doubly phosphorylated 20,000-dalton light chain from turkey gizzard myosin was hydrolyzed with alpha-chymotrypsin and the phosphorylated peptide was isolated by reverse phase chromatography. Following amino acid analyses and partial sequence determinations the second site of phosphorylation is shown to be threonine 18. This site is distinct from the threonine residue phosphorylated by protein kinase C. The time courses of phosphorylation of serine 19 and threonine 18 in isolated light chains follow a single exponential indicating a random process, although the phosphorylation rates differ considerably. The values of kcat/Km for serine 19 and threonine 18 for isolated light chains are 550 and 0.2 min-1 microM-1, respectively. With intact myosin, phosphorylation of serine 19 is biphasic; kcat/Km values are 22.5 and 7.5 min-1 microM-1 for the fast and slow phases, respectively. In contrast, phosphorylation of threonine 18 in intact myosin is a random, but markedly slower process, kcat/Km = 0.44 min-1 microM-1. Dephosphorylation of doubly phosphorylated myosin (approximately 4 mol of phosphate/mol of myosin) and isolated light chains (approximately 2 mol of phosphate/mol of light chain) follows a random process and dephosphorylation of the serine 19 and threonine 18 sites occurs at similar rates.     

Substrate based inhibitors of smooth muscle myosin light chain kinase.

Activation of myosin light chain kinase is a prerequisite for smooth muscle activation. In this study, short peptide analogs of the phosphorylation site of the myosin light chain were studied for their effects on several contractile protein systems. The peptides inhibited phosphorylation of isolated ventricular and smooth muscle myosin light chains by smooth muscle myosin light chain kinase, but they were only weak inhibitors of phosphorylation of intact myosin and actomyosin. The peptides were also unable to block force development or myosin light chain phosphorylation in glycerol permeabilized fibers of swine carotid media. Apparently, the association of the myosin light chain with myosin changes its conformation such that substrate analogs which are potent inhibitors of the phosphorylation of isolated myosin light chains by myosin light chain kinase are ineffective at blocking phosphorylation of the intact molecule.     

Phosphorylation of brush border myosin by brush border calmodulin-dependent myosin heavy and light chain kinases

Calmodulin-dependent myosin light chain kinase isolated from chicken intestinal brush border phosphorylates brush border myosin at an apparently single serine identical to that phosphorylated by smooth muscle myosin light chain kinase. Phosphorylation to 1.8 mol phosphate/mol myosin activated the myosin actin-activated ATPase about 10-fold, to about 50 nmol/min per mg. Myosin phosphorylated on its light chains could then be further phosphorylated to a total of 3.2 mol phosphate per mol by brush border calmodulin-dependent heavy chain kinase. Heavy chain phosphorylation did not alter the actin-activated ATPase of either myosin prephosphorylated on its light chains or of unphosphorylated myosin.     

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 Km and kcat and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys4 with Asp reduced actin binding and increased Km and kcat 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 Km for actin and the kcat 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.