Probing the structure of Escherichia coli catalase HPII

Changes in Val169, 8 A from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity. Changes in Asp181, 12 A from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gin, Ser, Asn or Ile are the substituted residues. It is proposed that an electrical potential difference exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 A distant. The second part of this study focused on, Ser234 and Glu530, situated 16 A and 20 A respectively, from active site heme in the main channel of HPII. These are not conserved residues and are present only in large subunit enzymes. A number of HPII variants were constructed and characterized at positions Ser234 and Glu530. The observed Km (H2O2 at Vmax/2) for the non-polar S234A and S234I variants are two to three fold higher than wild type showing that these variants have reduced affinity for the substrate, suggesting a need for hydrogen bonding at position 234. Another major focus of the work presented here, is to elucidate the role of the additional domains in large subunit catalases. HPH 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. 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. The truncated variants were purified on Superose12 FPLC column in amounts sufficient for crystallization trials. As expected, biochemical studies showed that the active site appears to be more accessible in N- & C-trunc variant than N-trunc and wild type HPII. (Abstract shortened by UMI.)...