Effects of substrates on the thermal stability of nuclear histone acetyltransferase.
Calf thymus nuclear histone acetyltransferase was found to have an optimal activity at about 30 degrees C with an energy of activation of 13.2 +/- 0.4 kcal/mol. The enzyme, however, is thermally unstable. At 38, 42, and 46 degrees C, the enzyme was inactivated with rate constants of 0.01, 0.033 and 0.097 min-1, respectively. High salt concentrations and histones accelerated the rate of thermal inactivation. Bovine serum albumin while alone having no effect on the inactivation process, decreased the exacerbation of heat inactivation caused by histones. Acetyl-CoA, on the other hand, protected the enzyme from heat denaturation. The acetyl-enzyme intermediate also underwent heat inactivated but at a much slower rate. The first order rate constant for the process at 42 degrees C decreased from 0.033 min-1 for the native enzyme to 0.013 min-1 for the acetyl-enzyme, corresponding to an increase in Arrhenius energy of inactivation from 55 kcal/mol to 82 kcal/mol. The distinct effects of acetyl-CoA and histones on the thermal stability of the enzyme suggest that interactions of the enzyme with these two substrates are different and thereby result in different enzyme substrate complexes. This observation further supports our previously proposed two-site ping-pong kinetic reaction mechanism which suggests that the two structurally and electrostatically different substrates bind to separate sites on the enzyme.
Sharpe
D J
DJ
Department of Biological Sciences, University of Lowell, MA 01854.
Wong
L J
LJ
D000105:Acetyl Coenzyme A / Q000494:pharmacology*; D000123:Acetyltransferases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects*; D051548:Histone Acetyltransferases; D006657:Histones / Q000494:pharmacology*; D007700:Kinetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013696:Temperature; D013950:Thymus Gland / Q000201:enzymology
D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D051548:Histone Acetyltransferases