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Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose-response curves.

ABSTRACT: A statistical treatment of steady-state enzyme kinetic measurements is described that allows for depletion of free substrate or free inhibitor concentrations owing to significant binding to the enzyme. V(max.), K(m) or K(i), enzyme concentration, the concentration of substrate or inhibitor required for a half-maximal effect and standard errors of these parameters can be calculated from dose-response measurements; the concentration of each component of the system may be estimated also. The statistically best values of the parameters are used to convert dose-response curves into convenient linear forms. The method is applied to dose-response measurements of hydroxyquinoline N-oxide inhibition of bacterial respiration and aminopterin inhibition of dihydrofolate reductase. Two FORTRAN programs for this method have been deposited as Supplementary Publication no. SUP 50019 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973) 131, 5.

SUBMITTER: Henderson PJ

PROVIDER: S-EPMC1165794 | biostudies-other | 1973 Sep

REPOSITORIES: biostudies-other

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Project description:Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.Significance of the studyPhosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

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Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose-response curves.

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