Catalase HPII from Escherichia coli exhibits enhanced resistance to denaturation

Catalase HPII from Escherichia coli is a homotetramer of 753 residue subuni ts. The multimer displays a number of unusual structural features, includin g interwoven subunits and a covalent bond between Tyr415 and His392, that w ould contribute to its rigidity and stability. As the temperature of a solu tion of HPII in 50 mM potassium phosphate buffer (pH 7) is raised from 50 t o 92 degrees C, the enzyme begins to lose activity at 78 degrees C and 50% inactivation has occurred at 83 degrees C, The inactivation is accompanied by absorbance changes at 280 and 407 nm and by changes in the CD spectrum c onsistent with small changes in secondary structure. The subunits in the di mer structure remain associated at 95 degrees C and show a significant leve l of dissociation only at 100 degrees C, The exceptional stability of the d imer association is consistent with the interwoven nature of the subunits a nd provides an explanation for the resistance to inactivation of the enzyme . For comparison, catalase-peroxidase HPI of E, coli and bovine liver catal ase are 50% inactivated at 53 and 56 degrees C, respectively. In 5.6 M urea , HPII exhibits a coincidence of inactivation, CD spectral change, and diss ociation of the dimer structure with a midpoint of 65 degrees C, The inacti ve mutant variants of HPII which fold poorly during synthesis and which lac k the Tyr-His covalent bond undergo spectral changes in the 78 to 84 degree s C range, revealing that the extra covalent linkage is not important in th e enhanced resistance to denaturation and that problems in the folding path way do not affect the ultimate stability of the folded structure.