Hydrogen transfer, substituted enzyme

The crystal structure of the cobalt-substituted enzyme was obtained with bicarbonate bound to the metal (Iverson et al. 2000). The structure shows Asn 202 and Gln75 hydrogen bonded to the metal-bound bicarbonate, suggestzing potential roles for these residues in either transition-state stabilization or orientation and polarization of CO2 for attack from the zinc-hydroxyl (Fig. 11.5). The crystal structure also shows three discrete conformations for Glu 84, suggesting a role for this residue in the transfer of protons out of the active site indeed, kinetic analyses of Glu 84 variants combined with chemical rescue experiments establish this residue as critical for proton transfer (Tripp and Ferry 2000). The location of Glu 62 adjacent to Glu 84 suggests a potential role in proton transfer as well. Although kinetic analyses of site-specific variants establish an essential role for Glu 62 in the CO2 hydration steps (Eqs. 11.3 and 11.4), the results were inconclusive regarding an additional role in proton transfer (Eqs. 11.5 and 11.6). 

An interesting and potentially useful observation in the mutational work is that the substitution of leucine for Hisl43o decreased the activity to 0.2% of wild type. This significant activity allowed the deuterium kinetic isotope effect for the reaction of [ 1- H2] propane-1,2-diol to be measured. The value obtained turned out to be 2 for ° cat, about one-fifth to one-sixth of that for wild-type enzyme. Substitution of alanine gave about 1.5% activity and a deuterium kinetic isotope effect of 5-6. It appears that the hydrogen transfer is not solely rate limiting in these variants. 

Lehn and coworkers have profitably employed tartaric acid-containing crown ethers as enzyme models. The rate of proton transfer to an ammonium-substituted pyridinium substrate from a tetra-l,4-dihydropyridine-substituted crown ether was considerably enhanced compared to that for a simple 1,4-dihydropyridine. The reaction showed first order kinetic data and was inhibited by potassium ions. Intramolecular proton transfer from receptor to substrate was thus inferred via the hydrogen bonded receptor-substrate complex shown in Figure 16a (78CC143). 

The PTA ligand has recently been employed as a water-soluble ligand in a variety of studies, including catalytic biphasic hydrogenation reactions,4,5 ligand-substitution reactions in metal clusters,6 and enzyme-mediated oxo-transfer processes.7... 

Eklund et al. suggested that the side chains of Ser 48 and His 51 act as a proton relay system to remove the proton from the alcohol, in step b of Eq. 15-7, leaving the transient zinc-bound alcoholate ion, which can then transfer a hydride ion to NAD+, in step c.52 The shaded hydrogen atom leaves as H+. The role of His 51 as a base is supported by studies of the inactivation of the horse liver enzyme by diethyl pyrocarbonate57 and by directed mutation of yeast and liver enzymes. When His 51 was substituted by Gin the pKa of 7 was abolished and the activity was decreased ten-fold.58... 

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