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Hydroperoxidase II of Escherichia coli exhibits enhanced resistance to proteolytic cleavage compared to other catalases

Hydroperoxidase II of Escherichia coli exhibits enhanced resistance to proteolytic cleavage compared to other catalases

Biochemistry 42(19): 5729-5735

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 resembles small 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 conditions of limited digestion, resulted in cleavage of 72-74 residues from the N-terminus of each subunit that created a homotetramer of 76 kDa subunits with 80% of wild-type activity. Longer treatment with proteinase K removed the C-terminal domain, producing a transient 59 kDa subunit which was subsequently cleaved into two fragments, 26 and 32 kDa. The tetrameric structure was retained despite this fragmentation, with four intermediates being observed between the 336 kDa native form and the 236 kDa fully truncated form corresponding to tetramers with a decreasing complement of C-termini (4, 3, 2, and 1). The truncated tetramers retained 80% of wild-type activity. The Tm for loss of activity during heating was decreased from 85 to 77 [degree]C by removal of the N-terminal sequence and to 59 [degree]C by removal of the C-terminal domain, revealing the importance of the C-terminal domain in enzyme stability. The sites of cleavage were determined by N- and C-terminal sequencing, and two were located on the surface of the tetramer with a third being exposed by removal of the C-terminal domain. Reprinted by permission of the publisher.

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Accession: 009872233

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PMID: 12741830

DOI: 10.1021/bi034208j

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