The ATPase mechanism of skeletal and smooth muscle acto-subfragment 1.

S S Rosenfeld,E W Taylor

doi:10.1016/s0021-9258(20)71298-6

Abstract

The transient and steady-state kinetic parameters for the ATPase cycle of the complex of skeletal muscle actin with smooth or skeletal muscle subfragment 1 (S-1) were determined over a wide range of actin concentrations from measurements of protein tryptophan fluorescence, the transient hydrolysis step, and the steady-state rate. The properties of the completely associated system were determined by using S-1 covalently cross-linking to actin. A four-state model was found to provide a good approximation to the kinetic and steady-state behavior: (sequence; see text) where T, D, and Pi refer to ATP, ADP, and inorganic phosphate, respectively. The formation of AM1T and M1T occurs by a rapid equilibrium binding of T followed by a very fast step. Actin is in rapid equilibrium with M1T and M2.DPi, with association constants K2 and K4. The three rate constants k1, k-1, and k5 were obtained by fitting observed rate constants from transient state measurements and VM to the model using values of k3 and k-3 determined for S-1 alone. To fit the data for skeletal or smooth muscle acto-S-1, the calculated rate constant of the hydrolysis step k1 and the equilibrium constant K1 had to be 2-3 times smaller than the corresponding parameters (k3, K3) for S-1. The calculated effective rate constant for product release must be large for striated muscle (85 s-1 at 20 degrees C, low ionic strength) and relatively small for smooth muscle (3 s-1). The difference in the actin-concentration dependence of association and of steady-state ATPase activity was predicted correctly from the rate constants fitted to the transient evidence. While the proposed mechanism does not exclude the possibility of additional ATP or product intermediate states, the properties of such states cannot be deduced from the kinetic evidence.

Highlights

The transient and steady-state kinetic parameters nism of mechano-chemical coupling in muscle andother for theATPase cycle of the complex of skeletal muscle motile systems
Necessary to determine the number of important intermediate states, the dominant pathway in the more complex scheme, and therelation of this where T, D, and Pi refer to ATP, ADP, and inorganic pathway to thecross-bridge cycle
Both experiments gave an amplitude of the phosphate burst of 0.6 f 0.05 and the rate constant s-1 of the hydrolysis step was 125 s-' for in approximate agreement with the rate measured by fluorescence,and 150S" for acto-S-1

Summary

The kinetic schemebemay expanded by introducing further

A t this ionic strength, the acto-S-1 will be almost completely dissociated by ATP in the initial mixing, and the hydrolysis reaction should be essentially the same as that for S-1alone Both experiments gave an amplitude of the phosphate burst of 0.6 f 0.05 and the rate constant s-1 of the hydrolysis step was 125 s-' for in approximate agreement with the rate measured by fluorescence,and 150S" for acto-S-1. Steady-state rate measurements with myosin in large excess over actinare described under "Results." A simple expression for the dependence of the rate of hydrolysis per actin residue on the myosin concentration cannotbe givenbecause the rateequations are nonlinear. The calculations indicated that a 50-to-1 ratio of myosin to actin would givea V M close to thevalue for an infinite ratio

RESULTS

Ea o

ASSOCIATION TIME

DISCUSSION