Catalase HPII from Escherichia coli Exhibits Enhanced Resistance to Denaturation

• Published in: Biochemistry (Easton) 1999-03, Vol.38 (13), p.3895-3901
• Main Authors: Switala, Jacek, O'Neil, Joe O, Loewen, Peter C
• Format: Article
• Language: English
• Subjects: Catalase - chemistry; Catalase - genetics; Catalase - metabolism; Circular Dichroism; Enzyme Activation - genetics; Enzyme Stability - genetics; Escherichia coli; Escherichia coli - enzymology; Guanidine; Hot Temperature; Models, Molecular; Mutagenesis, Site-Directed; Protein Denaturation; Sodium Dodecyl Sulfate; Urea
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982863z

Catalase HPII from Escherichia coli is a homotetramer of 753 residue subunits. The multimer displays a number of unusual structural features, including interwoven subunits and a covalent bond between Tyr415 and His392, that would contribute to its rigidity and stability. As the temperature of a solution of HPII in 50 mM potassium phosphate buffer (pH 7) is raised from 50 to 92 °C, the enzyme begins to lose activity at 78 °C and 50% inactivation has occurred at 83 °C. The inactivation is accompanied by absorbance changes at 280 and 407 nm and by changes in the CD spectrum consistent with small changes in secondary structure. The subunits in the dimer structure remain associated at 95 °C and show a significant level of dissociation only at 100 °C. The exceptional stability of the dimer association is consistent with the interwoven nature of the subunits and provides an explanation for the resistance to inactivation of the enzyme. For comparison, catalase-peroxidase HPI of E. coli and bovine liver catalase are 50% inactivated at 53 and 56 °C, respectively. In 5.6 M urea, HPII exhibits a coincidence of inactivation, CD spectral change, and dissociation of the dimer structure with a midpoint of 65 °C. The inactive mutant variants of HPII which fold poorly during synthesis and which lack the Tyr−His covalent bond undergo spectral changes in the 78 to 84 °C range, revealing that the extra covalent linkage is not important in the enhanced resistance to denaturation and that problems in the folding pathway do not affect the ultimate stability of the folded structure.