The stability curve of bovine adenosine deaminase is bimodal.
We have determined the stability curve of bovine adenosine deaminase via titrations with guanidine hydrochloride at pH = 6.3 from 5 to 65 degrees C. The data indicate that the enzyme undergoes an abrupt conformational transition at approximate, equals 29 degrees C, a finding supported by a temperature scan of the intrinsic enzyme fluorescence emission. Analysis of the data above and below this temperature with the modified Gibbs-Helmholtz equation allows for complete description of the equilibrium unfolding thermodynamics for either enzyme conformation. The high-temperature form of the enzyme is described by DeltaH degrees = 648 +/- 37 kJ/mole, DeltaC(P) = 23.2 +/- 2.5 kJ/mole-K, and a heat denaturational temperature T(h)(dn) = 72.5 +/- 0.9 degrees C. The low-temperature form is described by DeltaH degrees = 1284 +/- 47 kJ/mole, DeltaCP = 73.2 +/- 4.9 kJ/mole-K, and T(h)(dn) = 32.6 +/- 0.6 degrees C. Further thermodynamic analysis of the conformations that predominate at 38.3 degrees C, the bovine normal body temperature, and at 4 degrees C, where the crystals for x-ray structural analyses were formed, suggest that the stability of either form is due to favorable amino acid side chain nonpolar interactions with these interactions being much more optimized in the low-temperature conformation. We therefore conclude that the structure as determined by x-ray crystallographic methods cannot be the physiological structure. The data also suggest that the general calculation of enzyme stability curves from the extrapolation of heat denaturation data may inaccurately represent the enzyme stability as a low-temperature, nondenaturational transition is assumed not to exist. Further consequences in terms of general enzyme catalysis are also discussed.
Anderson
Esther
E
Department of Chemistry and Physics, Texas Woman's University, Denton 76204-5859, USA.
Britt
B Mark
BM
D000243:Adenosine Deaminase / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics
D000243:Adenosine Deaminase; D019791:Guanidine