Kinetic characterization of reductively methylated myosin subfragment 1.
Reductive methylation of myosin-S1 converts 97% of lysine residues in native myosin-S1 to dimethyllysine without detectable modification of other amino acid side chains. RM-S1 is catalytically active, although the rate and equilibrium constants of many of the steps of the actomyosin ATP hydrolysis mechanism have been altered. The steady-state rate of MgATP hydrolysis by modified myosin-S1 (RM-S1) is increased 4-5-fold in the absence of actin. However, the maximum steady-state rate of RM-S1 at saturating actin, 0.59 s-1, is less than one-tenth that observed for the unmodified protein, 7.4 s-1 (5 mM MOPS, 2 mM MgCl2, pH 7, 20 degrees C). Under single-turnover conditions [S1] > [ATP], the observed rate of ATP hydrolysis by RM-S1 is fit by a single exponential that is no more than twice the steady-state rate, which indicates that the bond splitting state is at least partially rate limiting for RM-S1. Although a small decrease in intrinsic tryptophan fluorescence is observed upon ATP binding to RM-acto-S1, the large and rapid fluorescence enhancement produced by aza-ATP binding to RM-acto-S1 is nearly normal. ATP binds and dissociates modified RM-S1 from actin with a second-order rate constant that is more than twice that observed for control S1. The changes in the kinetic mechanism produced by reductive methylation of lysine are qualitatively and quantitatively similar to the changes that are induced by either SH1 modification or substrate analogues such as GTP.
White
H D
HD
Department of Biochemistry, East Virginia Medical School, Norfolk 23501.
Rayment
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D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D002645:Chickens; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D015879:Myosin Subfragments; D000205:Actomyosin; D009218:Myosins; D008239:Lysine