Differential scanning calorimetric study of the thermal denaturation of aspartate transcarbamoylase of Escherichia coli.
The thermal denaturation of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of high-sensitivity differential scanning calorimetry (DSC). As previously reported [Vickers, K.P., Donovan, J.W., & Schachman, H.K. (1978) J. Biol. Chem. 253, 8493-8498], the denaturational endotherm consists of two peaks, the lower of which is due to denaturation of the three regulatory, r2, subunits while the upper involves the two catalytic, c3, subunits. The temperature of maximal excess apparent specific heat, tm, of the lower peak is raised from the value of 51.4 degrees C for the isolated subunit to 66.8 degrees C as a result of subunit interactions, whereas tm for the c3 peak is essentially the same in the isolated subunit and in the holoenzyme, indicating that the denatured r2 subunits do not interact with the c3 subunits. The total specific denaturational enthalpy for c6r6, 4.83 +/- 0.16 cal g-1, is significantly larger than the weighted mean, 4.08 cal g-1, of the enthalpies for c3 and r2. The fact that no endotherm is observed when previously scanned protein is rescanned indicates that the denaturation is irreversible, as is also the case with the r2 and c3 subunits. Empirical justification for analyzing the data in terms of equilibrium thermodynamics is cited. The observed DSC curves can be expressed within experimental uncertainty as the sum of five sequential two-state steps. The value of t 1/2, the temperature of half-completion, for each step increases with increasing protein concentration, indicating that some dissociation of the protein takes place during denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)
Edge
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Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06457.
Allewell
N M
NM
Sturtevant
J M
JM
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D011489:Protein Denaturation
D001221:Aspartate Carbamoyltransferase