This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Co(II)-substituted beta-carbonic anhydrase from H. influenzae has recently been produced. The visible spectrum of the Co(II) enzyme is sensitive to allosteric state of the enzyme. X-ray structural analysis of this enzyme is important to (1) demonstrate that the Co(II) enzyme is isostructural with the wild type, Zn(II) enzyme, and (2) determine if bicarbonate ion (both a substrate and allosteric effector) binds directly to the metal ion, to the allosteric site, or both. Two samples of this enzyme have been crystallized, with and without bicarbonate ion ligand present. The crystals, which are 0.2-0.3 mm in size, are an improved form of monoclinic crystals we collected unsuccessful data on last April. While we have not yet screened these crystals, we would expect them to diffract as well as our prior sample (approx. 2.0 A) Variant R64A of H. influenzae carbonic anhydrase has been prepared to explore the role of the allosteric binding site in this enzyme. Arg64 is believed to be a critical residue in bicarbonate ion binding to the allosteric site. X-ray structural analysis of this enzyme is important to (1) determine which of the two allosteric states the enzyme has adopted as a result of this mutation, and (2) whether or not bicarbonate ion can bind to the partially modified allosteric binding site. Two samples of this enzyme have been crystallized, with and without bicarbonate ion present. The crystals are 0.2-0.4 mm in size and clearly tetragonal (most likely P41212) , similar to crystals of other variants we have prepared. We have not yet screened these crystals, but expect them to diffract well based on past experience. Altogether we need to collect 4 complete datasets, two for each protein sample described above. Structures will be solved by molecular replacement, using the wild-type enzyme or one of our existing variant protein structures. The experimental data collection and reduction should be straightforward. In addition, there is the possibility that one or more undergraduate students could assist in data collection and analysis, depending on scheduling.
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