Myosin activation kinetics

Kurzawa-Goertz, S. E., Perreault-Micale, C. L., Trybus, K. M., Szent-Gyorgyi, A. G., and Geeves, M. A. (1998). Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of SI isoforms. 

Szent-Gyorgyi further showed that the viscosity of an actomyosin solution was lowered by the addition of ATP, indicating that ATP decreases myosin s affinity for actin. Kinetic studies demonstrated that myosin ATPase activity was increased substantially by actin. (For this reason, Szent-Gyorgyi gave the name actin to the thin filament protein.) The ATPase turnover number of pure myosin is 0.05/sec. In the presence of actin, however, the turnover number increases to about 10/sec, a number more like that of intact muscle fibers. 

The importance of Ser-19 phosphorylation for actin-activated smooth muscle myosin MgATPase activity and for contraction of smooth muscle stimulated research to find out which amino acids, surrounding the serine, are required for the phosphorylation. Using synthetic peptide analogs of the native phosphorylation site comprising residues from Lys-11 to Ala-23, it was shown that Arg-16 had a strong influence on the kinetics of peptide phosphorylation (Kemp and Pearson, 1985). The location of Arg-16 in relation to Ser-19, as well as the distance between Arg-13 and Arg-16, was found to be important. Placement of Arg-16 at position 15 caused a complete switch in specificity from the natural Ser-19 phosphorylation site to Thr-18. Increasing the number of alanine residues between Arg-13 and Arg-16 in the model peptide also influenced the kinetics and site specificity of peptide phosphorylation. On the carboxyl side of Ser-19, Val-21 and Phe-22 influenced the of peptide phosphorylation, whereas Ala-23 was found not to be essential (Pearson et ah, 1986). 

Kinetic studies have shown that filament initiation is more difficult than subsequent elongation (Cross et al., 1991). In a system where assembly-disassembly might play a large role, for example, in nonmuscle vertebrate cells, this property predicts that the rate at which monomers become available for polymerization could alter both the number and length of myosin filaments that are formed. Thus control of kinase activity, which controls the number of assembly competent extended monomers, could be a factor in determining subsequent polymerization. 

Another set of smooth muscle heavy chain isoforms that have either the insertion or omission of 7 amino acids in the head domain near the ATP-binding region have been identified (Babij 1993, Kelly et al 1993, White et al 1993). The insertion appears to be present in visceral smooth muscle tissues but is absent in tonic vascular smooth muscle, and appears to confer functional differences in the kinetic properties of smooth muscle myosins from visceral smooth muscle. There is evidence from studies in the motility assay that the visceral smooth muscle isoform has higher ATPase activity and moves actin filaments faster than the vascular isoform (Kelly et al 1993, Rovner et al 1997). However, because the head domain isoforms may be associated with different tail domain isoforms in different tissues, as well as with different light chain isoforms, it has been difficult to establish causal relationships between myosin heavy chain isoforms and myosin kinetics with certainty. (Reviewed by Somlyo 1993, Murphy et al 1997). 

Klemt P, Peiper U, Speden RN, Zilker F (1981) The kinetics of post-vibration tension recovery of the isolated rat portal vein. J Physiol (Lond) 312 281-296 Kobayashi H, Inoue A, Mikawa T, Kuwayama H, Hotta Y, Masaki T, Ebashi S (1992) Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity. J Biochem (Tokyo) 112 786-791 Kokubu N, Satoh M, Takayanagi I (1995) Involvement of botulinum C3-sensitive GTP-binding proteins in ai-adrenoceptor subtypes mediating Ca -sensitization. Eur J Pharmacol 290 19-27... 

This kinetic limit is clearly appropriate for muscle. The value <7 = 0.015 corresponds to AA5 = A5 (0) Af (a) lOkJ/mol. Myosin V is another example of a kinetically controlled motor. The difference in activation barriers for the and kinetic processes are 40 kJ/mol, as can be seen from the energy landscapes for the functional and non-functional paths [23]. Both of these values are indeed significantly smaller than the A/i for ATP hydrolysis. Forcing an ATP-driven kinetically controlled motor backwards causes enhanced ATP hydrolysis, not synthesis, a behavior predicted by Astumian and Bier [10]. 

The time course of the labeling of myosin by Co-(phen)-ATP (Fig. 1) occurs in two phases at first ATPase activity (Ca +-, EDTA-, and actin-dependent activities) are enhanced, and in the second phase the activities are abolished in a pseudo-first-order process. In the cases of the myosin subfragments— double-headed heavy meromyosin (HMM) and single-headed subfragment 1 (S-1)—no enhancement phase is observed and the labeling occurs as a pseudo-first-order inactivation process. The kinetic parameters of the Co-(phen)-ATP affinity labeling of myosin and its subfragments are summarized in Table II. 

Without any doubt, one of the most extensively sttidied enzyme reaction by transient kinetics is the ATPase activity of the subfragment-1 (SI) of myosin. This proteolytic product constitutes the heads of the myosin molecule, which has, in addition, a hinge and a tail component (see figure 4.11). SI contains the fully competent enzymatic site and also the site for attachment to actin, the thin filament of the myofibril. The interaction between SI and actin, discussed in section 6.4, is the key process in muscle contraction, and is controlled by the steps in ATP hydrolysis (Geeves et al., 1984 Geeves, 1991). The reciprocal relation between the modtilation of the... 

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