Thermal stability of phosphoribosyladenosine triphosphate synthetase as reflected in its circular dichroism and activity properties. Effect of inhibitors

Abstract 1. 1. The circular dichroism (CD) of phosphoribosyladenosine triphosphate: pyrophosphate phosphoribosyltransferase (phosphoribosyladenosine triphosphate synthetase) from Escherichia coli in the far ultraviolet region has been investigated. The CD spectrum of the enzyme is characterized by CD bands centering at 223, 210 and 193 nm, with [θ]λ = −14800, −14000 and +18000 degrees·cm 2 · mole −1 , respectively. A negligible dichroism in the near ultraviolet region has been observed. The analysis of the CD spectra based upon the published model parameters for α-, β- and random structures suggests the presence of approx. 33% α-helix, 20–30% β-structure, with the remaining portion of the enzyme existing in an unordered conformation. Histidine and AMP, inhibitors of phosphoribosyladenosine triphosphate synthetase, were found to have little or no effect on the CD spectra of the enzyme. 2. 2. The inhibitors histidine and AMP both protect the ordered structure against unfolding or thermodenaturation, with histidine being the more effective of the two. 3. 3. Upon heating to about 50°C for 10 min the negative ellipticity at 223 nm decreased irreversibly to a few percent of the value for the native enzyme, whereas only a slight reduction in the catalytic activity was observed. Thus, unfolding in parts of the native structure need not correspondingly influence the catalytic capability of the enzyme. 4. 4. Both histidine and AMP stabilized the enzyme with respect to thermal inactivation.