Abstract
Native horseradish peroxidase and several forms with mutations in the distal heme pocket (His42Leu, His42Arg, and Arg38Leu) have been expressed in Escherichia coli. These enzymes have been purified and analyzed in terms of the room temperature recombination rate of carbon monoxide and cyanide after photolysis of the reduced forms. The recombinant wild-type ferrous form exhibited monophasic recombination of carbon monoxide with an observed bimolecular rate constant at pH 8.5 of 4.4 x 10(3) M-1 s-1 which is essentially the same as the natural glycosylated form. This recombination rate constant increases in the mutants in the order WT < H42R < H42L << R38L. The value for R38L (5 x 10(6) M-1 s-1) is increased by 3 orders of magnitude relative to the wild-type and is similar to that for human hemoglobin [Mims et al. (1983) J. Biol. Chem. 258, 14219-14232]. Cyanide recombination with the wild-type ferrous form at room temperature is biphasic at pH 6.5 but becomes more monophasic at pH 8.5, again similar to the behavior of the natural glycosylated form, although the Fe(2+)-cyano form of the recombinant enzyme appears to be more unstable at high pH. None of the mutant forms were able to bind cyanide in the ferrous state to any significant extent (Kdiss > 250 mM) when cyanide was added at a concentration (10-20 mM) sufficient to almost saturate the wild-type form (Kdiss approximately equal to 1 mM at pH 7). This behavior contrasts with that of the oxidized forms of the mutants where increases in cyanide dissociation constants are smaller ( < 25 times).(ABSTRACT TRUNCATED AT 250 WORDS)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.