Hydroperoxidase II of Escherichia coli Exhibits Enhanced Resistance to Proteolytic Cleavage Compared to Other Catalases
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- 作者：Prashen Chelikani ; Lynda J. Donald ; Harry W. Duckworth ; and Peter C. Loewen
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：May 20, 2003
- 年：2003
- 卷：42
- 期：19
- 页码：5729 - 5735
- 全文大小：430K
- 年卷期：v.42,no.19(May 20, 2003)
- ISSN：1520-4995
文摘
Catalase (hydroperoxidase) HPII of Escherichia coli is the largest catalase so far characterized,existing as a homotetramer of 84 kDa subunits. Each subunit has a core structure that closely resemblessmall subunit catalases, supplemented with an extended N-terminal sequence and compact flavodoxin-like C-terminal domain. Treatment of HPII with trypsin, chymotrypsin, or proteinase K, under conditionsof limited digestion, resulted in cleavage of 72-74 residues from the N-terminus of each subunit thatcreated a homotetramer of 76 kDa subunits with 80% of wild-type activity. Longer treatment with proteinaseK removed the C-terminal domain, producing a transient 59 kDa subunit which was subsequently cleavedinto two fragments, 26 and 32 kDa. The tetrameric structure was retained despite this fragmentation, withfour intermediates being observed between the 336 kDa native form and the 236 kDa fully truncatedform corresponding to tetramers with a decreasing complement of C-termini (4, 3, 2, and 1). The truncatedtetramers retained 80% of wild-type activity. The Tm for loss of activity during heating was decreasedfrom 85 to 77 
C by removal of the N-terminal sequence and to 59 C by removal of the C-terminaldomain, revealing the importance of the C-terminal domain in enzyme stability. The sites of cleavagewere determined by N- and C-terminal sequencing, and two were located on the surface of the tetramerwith a third being exposed by removal of the C-terminal domain.
C by removal of the N-terminal sequence and to 59 C by removal of the C-terminaldomain, revealing the importance of the C-terminal domain in enzyme stability. The sites of cleavagewere determined by N- and C-terminal sequencing, and two were located on the surface of the tetramerwith a third being exposed by removal of the C-terminal domain.