Water Networks in Fast Proton Transfer during Catalysis by Human Carbonic Anhydrase II

Abstract

Variants of human carbonic anhydrase II (HCA II) with amino-acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and 18O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing the replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for proton transfer from His64 to the zinc-bound hydroxide in catalysis were 4 μs-1 and 9 μs-1 for Y7F and Y7F-N67Q, respectively, compared with a value of 0.8 μs-1 for wild-type HCA II. These higher values observed for Y7F and Y7F-N67Q HCA II could not be explained by differences in the values of the pKa of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with reduced branching in the networks of hydrogen-bonded water molecules between the proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5 – 1.6 Å resolution. Moreover, Y7F-N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and Nδ of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.

Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense

“…This residue is located in the enzyme active site and is connected to the zinc-bound water molecule through a well ordered hydrogen bonded water network. [6][7][8][9] In the majority of human α-CA isoforms, this residue is a histidine (e.g., His64 in hCA II, IV, VII, IX, etc). 2,5 For a long time α-CAs were studied mainly for their medicinal chemistry applications as drug targets; only recently, in parallel to these uses, these enzymes have seen an increasing interest in their employment as biocatalysts for carbon dioxide sequestration processes and biofuel production.…”

Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure–Activity Relationships of Glucosyl-Based Sulfamates

“…Furthermore, binding of 623 causes an almost complete displacement of the ordered water network in the active site that has been determined important for catalysis. 26,27 The only water present in the enzymes active site is observed coordinated between the hydroxyl of the side chain of Thr200 and the NH of the sulfonamide linker of 623 (Figure 2A). The "tail" region of 623, comprising the sulfamate-linked acetylated glucose moiety shows limited interaction with the hCA IX-mimic active site.…”

Philosophy of voltage-gated proton channels

“…In a linear water chain, the proton can hop forward or backward along the file, but not sideways or up and down. Moreover, a protein may construct a channel that optimizes the positions of the waters for rapid proton transfer [99]; branching in a hydrogen-bonded water network slows proton conduction [105].…”