Deepa Bhatt

Proton transfer in a Thr200His mutant of human carbonic anhydrase II

Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc‐bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A‐T200H HCAII), effectively relocating the proton shuttle residue ∼ 2 Å closer to the zinc‐bound hydroxide compared to wild type HCA II. The crystal structure of H64A‐T200H HCA II at 1.8 Å resolution shows the side chain of His200 directly hydrogen‐bonded with the zinc‐bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration‐dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A‐T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc‐bound hydroxide, in analogy with proton transfer involving His64 in wild‐type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc‐bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen‐bonded to the zinc‐bound hydroxide, can adopt the correct distance geometry to support proton transfer. Proteins 2005.

Mutagenesis studies of the F1F0 ATP synthase b subunit membrane domain

A homodimer of b subunits constitutes the peripheral stalk linking the F1 and F0 sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F1F0 ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F1F0 ATPase and no detectable ATP-driven proton pumping activity. Expression of the bN2A,T6A,Q10A subunit was also oxidative phosphorylation deficient, but the bN2A,T6A,Q10A protein was incorporated into an F1F0 complex. Single amino acid substitutions had minimal reductions in F1F0 ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F0, and that these interactions are strongest on the periplasmic side of the bilayer.

Correlation of kinetic data with crystallographic structures of Aspartic Proteinase

Ben M. Dunn, Kohei Oda, Marina Bukhtiyarova, Deepa Bhatt, Chetana Rao-Naik, W. Todd Lowther, Paula E. Scarborough, Brian M. Beyer, Jennifer Westling, John Kay, Nora Cronin and Tom Blundell University of Florida College of Medicine, Gainesville, FL, 32610-0245 USA, Kyoto Institute of Technology, Kyoto, Japan, College of Molecular and Medical Biosciences, University of Wales, College of Cardiff, Cardiff, Wales, UK, Department of Biochemistry, University of Cambridge, Cambridge, UK.

Construction of Chimeric Enzymes to Probe Subsite Contributions to Catalytic Specificity

Aspartic proteinases are characterized by a high degree of sequence as well as structural homology.1 The subtle differences in specificity of these enzymes arise from interactions between the side chains of the ligand and the solvent exposed amino acids in the subsites that line the active site cleft. Studies using site-directed mutagenesis have suggested that although specific residues may contribute towards enzymatic efficiency, long range electrostatic interactions may also be involved.2,3 These interactions may arise from the interdependency between subsites that are present on the two domains. The subsites present within the N-terminal domain include S5, S3, S1, and the S2′ while those contributed by the C-terminal domain include S4, S2, S1′ and S3′.