Biphasic steady-state kinetics of myosin adenosine triphosphatase. Evidence for a substrate effector site.
The steady-state kinetics of the K+, Ca2+, and Mg2+-activated adenosine triphosphatase (ATPase) activities of rabbit skeletal myosin were investigated in the substrate concentration range from 0.05 microM to 5 mM and found not to follow Michaelis-Menten kinetics but rather to display biphasic behavior. The Ca2+-ATPase activity of myosin chymotryptic subfragment-1 (S-1), which has only one active site, also exhibits biphasic kinetics, thus excluding the possibility that the biphasic behavior is caused by negative cooperativity between the two active sites of myosin. Myosin K+ and Mg2+-ATPase are both activated by 5'-adenyl methylenediphosphonate (AdoPP[CH2]P) in a competitive manner at high substrate concentrations; i.e. the maximal velocity observed at high substrate concentrations is independent of the AdoPP[CH2]P concentration. This result provides evidence for substrate activation via binding to a regulatory site. Pyrophosphate inhibits myosin ATPase in a competitive manner at low substrate concentrations and in an uncompetitive manner at high substrate concentrations, with the uncompetitive Ki being smaller than the competitive Ki; i.e. pyrophosphate binds more tightly to the effector site than to the active site.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D027682:Cation Transport Proteins; D055598:Chemical Phenomena; D002621:Chemistry; D011756:Diphosphates; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits
D027682:Cation Transport Proteins; D011756:Diphosphates; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C022761:potassium transporting ATPase; D009218:Myosins; D000252:Calcium-Transporting ATPases; C002630:alpha,beta-methyleneadenosine 5'-triphosphate