Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase

Chingkuang Tu, Minzhang Qian, Haiqian An, Nina R. Wadhwa, David Duda, Craig Yoshioka, Yashash Pathak, Robert McKenna, Philip J. Laipis, David N. Silverman

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10 Scopus citations

Abstract

We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His7 in HCA III. Using an 18O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His7 and His64 within the active site cavity, shows the conformation of the side chain of His64 moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His7. This suggests a cause of decreased proton transfer in catalysis.

Original languageEnglish (US)
Pages (from-to)38870-38876
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number41
DOIs
StatePublished - Oct 11 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Tu, C., Qian, M., An, H., Wadhwa, N. R., Duda, D., Yoshioka, C., Pathak, Y., McKenna, R., Laipis, P. J., & Silverman, D. N. (2002). Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase. Journal of Biological Chemistry, 277(41), 38870-38876. https://doi.org/10.1074/jbc.M205791200