Codex @codex  0

The cation obtained by protonation or suitable N-alkylation of imidazole.

The compound imidazole resembles the metal-coordinating part of the histidine side chain and competes for binding to the nickel sites in immobilized metal affinity chromatography. At the lower concentration in the wash, it helps displace weakly or non-specifically bound proteins while the multiple contacts of the histidine tag retain the desired enzyme. At the higher elution concentration, competition is strong enough to displace the tagged enzyme. Low imidazole improves washing; high imidazole elutes the target. Remove or match the elution-buffer components before interpreting downstream assays, and verify purity independently of total protein concentration.

His238 contributes strongly to turnover, with a plausible acid/base or electrostatic role near the attacking water. Replacing its imidazole by the methyl side chain of alanine removes both proton-transfer capacity and a positioned polar group, so the large fall in turnover number fits impaired chemical catalysis. A candidate role is acceptance of a proton from zinc-bound water, or stabilization and subsequent proton shuttling around the attacking oxygen.

Little change in the Michaelis constant suggests that the major kinetic defect is not a gross loss of substrate recognition, but $K_M$ is not generally a binding dissociation constant: $K_M=(k_{-1}+k_{\mathrm{cat}})/k_1$ for the elementary scheme. Nor does reduced $k_{\mathrm{cat}}$ uniquely prove that His238 is the initial general base. Purified-protein controls should check fold, zinc occupancy and active concentration. Importantly, primary mouse-enzyme experiments found that His238 mutants could still hydrate a substrate analogue, directly illustrating this mechanistic limitation.^[ref]

Each imidazole already has one nitrogen substituted. Its remaining pyridine-like nitrogen attacks a benzylic carbon bearing chlorine in an $S_{\mathrm{N}}2$ reaction, expelling chloride and producing an imidazolium center. Repetition at the other end closes the ring. The expected product is therefore a dicationic bis-imidazolium macrocycle, in which two meta-xylylene bridges connect the two imidazolium rings. Both nitrogens of each imidazolium are alkylated; the aryl rings remain intact.

A compact way to specify its connectivity is to traverse the ring:Here each indicated imidazolium connects its two substituents through its two different ring nitrogen atoms, rather than through a single nitrogen.

chloride acts as an anion template: association with the positively charged intermediates and the inward-facing imidazolium CH donors brings the reacting ends together, favoring cyclization over intermolecular oligomerization. Tetrabutylammonium is a spectator counterion, not a covalent part of the product. The two substitutions produce two chloride counterions for the dication; template chloride can also occupy the cavity, with the remaining ions in solution. The schematic below specifies connectivity and charge without reproducing the source drawing.