Reversal of the Hydrogen Bond to Zinc Ligand Histidine-119 Dramatically Diminishes Catalysis and Enhances Metal Equilibration Kinetics in Carbonic Anhydrase II

Abstract

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.

The Activity and Cofactor Preferences of N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB) Change Depending on Environmental Conditions

“…It should be pointed out that the dissociation of Zn(II) from MshB does not go to an end point of zero. Similar findings have been reported for carboxypeptidase A (34,35), carbonic anhydrase (36), and, to some extent, LpxC (27) and may suggest that a fraction of the active site Zn(II) dissociates more rapidly. Although the association rate constant for Zn(II) with MshB approaches that of a diffusion-controlled process (2.8 ϫ 10 7 M Ϫ1 s Ϫ1 ), the association .…”

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

“…This design was particularly challenging, because it required the burial of six polar ionizable groups in the hydrophobic core, which were stabilized via second-shell hydrogen bonded interactions. Such second-shell interactions may prove to be essential for highaffinity binding and fine tuning of function (77,78) and have not been considered in previous attempts to design metalloproteins. The success described here in the design and structure elucidation of a complex metalloprotein has significant implications for the evolution of natural proteins, the understanding of dimetal centers, and the automated design of metalloproteins.…”

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

“…Of the three available coordinate sets for R. sphaeroides cytochrome c 2 (1CXA, 2CXB, and 1CXC), 1CXC was used because it is the most complete and determined at the highest resolution (1.6 Å). Missing atoms for residues 10,32,33,35,74,78,85,89,95,97,105, and 106 were added with Insight while using other two structures as guides. Both termini were charged, and both His 75 and His 111 were doubly protonated to satisfy intramolecular salt bridges, giving a total charge of 0.…”