Impact of Mutations on the Higher Order Structure and Activity of a Recombinant Uricase.

This study explores the structural and functional changes associated with a low-temperature thermal transition of 2 engineered bacterial uricase mutants. Uricase has a noncovalent homotetrameric structure, with 4 active sites located at the interface of subunits. Using differential scanning calorimetry, a low-temperature transition was identified at 42°C for mutant A and at 33°C for mutant B. This transition was stabilized by the uricase inhibitor, oxonic acid, suggesting a strong structural relationship to the active site. For mutant B, there was a reversible loss of enzymatic activity above the low-temperature transition. Spectroscopic measurements demonstrated that there was also a reversible loss of secondary and tertiary structures and an increase in surface hydrophobicity. However, the hydrophobic core environment and the tetrameric structure were not altered over the low-temperature transition suggesting that the changes occurred primarily at the surface of the enzyme. The protein became aggregation-prone at temperatures approaching the cluster of higher-temperature melting transitions at 84°C, indicating these transitions represent a global unfolding of the protein. Our findings shed light on the structural changes that affect the uricase mechanism of action and provide new insights into how enzyme therapeutic development may be approached.