No abstract

Carbonic anhydrase II (CAII) contains a conserved His3 zinc polyhedron which is essential for catalysis. Removal of any one of the His ligands by replacement with Ala decreases (approximately 10(5)-fold), but does not abolish, zinc binding and increases the rate constant for zinc dissociation. CAII variants with a His ligand substituted with Cys, Asp, or Glu bind zinc only approximately 10-fold better than a His2 zinc polyhedron in CAII. The large decrease in zinc affinity (approximately 5 kcal/mol) in these variants compared to the wild-type His3 site reflects mainly unfavorable compensatory protein structural rearrangements observed in the X-ray crystallographic structures of some of these CAII variants, described by Ippolito and Christianson (following paper in this issue). However, the zinc affinity of these sites is still higher than zinc polyhedra designed de novo. Substitution of the His zinc ligands with negatively charged amino acids both increases the pKa of the zinc-bound water by > or = 1.6 pH units, confirming that neutral ligands maintain the low zinc-water pKa, and decreases the pH-independent kcat/KM for ester hydrolysis (3-30-fold) and CO2 hydration (approximately 10(3)-10(5)-fold). Additionally, decreases in the dissociation constant (approximately approximately 10(2)-10(5)-fold) for the transition state analog acetazolamide correlate with the decreased catalytic efficiency and increased pKa of these CAII variants. These data indicate that the histidine ligands, although not essential for catalysis, are conserved to maximize electrostatic stabilization of both the ground-state zinc-hydroxide and the negatively charged transition state. These studies provide valuable insights into the functional consequences of engineering a catalytic zinc site in a metalloenzyme.

The murine agouti gene encodes for a novel 131 amino acid protein. The sequence includes a 22 residue putative secretion signal, an internal basic region, and a C-terminal domain containing 10 cysteines. Agouti has been found to antagonize the binding of certain pro-opiomelanocortin peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH), to the murine melanocortin-1 receptor (MC1-R). We report the purification of a secreted murine agouti to homogeneity by a two-step procedure from baculovirus-infected Trichoplusia ni (T. ni). The protein is glycosylated and exhibits competitive, high-affinity antagonism (Ki = 0.8 nM) versus alpha-MSH in cell-based assays employing B16F10 cells. Association state analysis by analytical ultracentrifugation reveals that agouti exists in a monomer--dimer plus aggregate equilibrium at low micromolar concentrations. Data from secondary structure studies indicate that the protein is highly stable to thermal denaturation. Enzymatic digestion to probe disulfide bond arrangement yielded a discrete C-terminal (Val 83-Cys 131) domain. The isolated highly cysteine-rich C-terminal domain retains alpha-MSH antagonism equipotent with mature agouti. This bioactive domain contains all 10 cysteines which exhibit sequence homology when aligned with several conotoxins.

Direct metal ligands to transition metals in metalloproteins exert a profound effect on protein-metal affinity and function. Indirect ligands, i.e., second-shell residues that hydrogen bond to direct metal ligands, typically exert more subtle effects on the chemical properties of the protein-metal complex. However, E117 of human carbonic anhydrase II (CAII), which is part of the E117-119-Zn(2+) triad, is a notable exception: E117-substituted CAIIs exhibit dramatically increased kinetics of zinc complexation, and the E117Q variant exhibits enormously diminished catalytic activity and sulfonamide affinity. The three-dimensional structures of zinc-bound and zinc-free E117Q CAII reveal no discrete structural changes in the active site that are responsible for enhanced zinc equilibration kinetics and decreased activity. Additionally, the structure of the acetazolamide complex is essentially identical to that of the wild-type enzyme despite the 10(4)-fold loss of enzyme-inhibitor affinity. We conclude, therefore, that the functional differences between E117Q and wild-type CAIIs arise from electrostatic and not structural differences in the active site. We propose that the E117Q substitution reverses the polarity of the residue 117-H119 hydrogen bond, thereby stabilizing H119 as a histidinate anion in the E117Q CAII holoenzyme. The additional negative charge in the first coordination sphere of the metal ion increases the pK(a) of the zinc-water ligand, destabilizes the transition state for CO(2) hydration, and facilitates the exchange of a zinc-histidine ligand with an additional water molecule by decreasing the stability of the tetrahedral zinc complex. These novel properties engineered into E117Q CAII facilitate the exploitation of CAII as a rapid and sensitive Zn(2+) biosensor.