Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromyosin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ.
Transient kinetic studies of Mg(2+)-dependent heavy-meromyosin ATPase (adenosine triphosphatase) were done by monitoring the release of both ADP and P(i) into the reaction medium by using linked assay systems. The release of P(i) was monitored by its quantitative transfer to ADP, with concomitant reduction of NAD(+) in the presence of d-glyceraldehyde 3-phosphate, d-glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. The dissociation rates of the products, ADP and P(i), from heavy meromyosin were shown to be faster than the rate-controlling process, which occurs after the initial bond cleavage of ATP. The chromophoric ATP analogue, 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (thioATP) was used as a substrate and spectral changes associated with a single turnover of heavy meromyosin could be assigned to elementary processes of the mechanism. It was shown that the dissociation rate of thioADP was not the rate-controlling process of the thioATPase, whose catalytic-centre activity was 7.6 times that of the ATPase at pH8. The dissociation rate of ADP from heavy meromyosin was measured by using thioATP as displacing agent and was found to be 2.3s(-1), which is about 50 times the catalytic-centre activity of the ATPase at pH8. Transient kinetic studies with chromophoric adenosine phosphate analogues have general application for kinases and ATPases both in characterizing the chemical states of the intermediates and in delineating the elementary processes of the enzyme mechanism.
D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D000818:Animals; D003124:Colorimetry; D007700:Kinetics; D008274:Magnesium; D008722:Methods; D009124:Muscle Proteins; D015879:Myosin Subfragments; D010710:Phosphates / Q000032:analysis*; D010770:Phosphotransferases; D011817:Rabbits; D013053:Spectrophotometry
D009124:Muscle Proteins; D015879:Myosin Subfragments; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000251:Adenosine Triphosphatases; D008274:Magnesium