Proton-relay system

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... 

Dworschack and Plapp presented a mechanism based on this proton relay system and this is shown in Scheme The alcohol or carbonyl groups are bonded to the zinc, as is the water molecule, giving a five-coordinate zinc. 

This enzyme, which is relatively stable under reaction conditions, will retain 70% of its activity after 10 days at pH 5 and 25°C. Although it is not yet commercially available, it has been overexpressed in E. coli, making large quantities easily accessible.68 The detailed mechanism of DERA has been determined based on the atomic structure (ca. 1.0 A) combined with site-directed mutagenesis, kinetic, and NMR studies136 (Scheme 5Alb). A proton relay system composed of Lys and Asp appears to activate a conserved active-site water that functions as the critical mediator for proton transfer in acid-base catalysis. 

Tapia and Eklund (1986) carried out a Monte Carlo simulation of the substrate channel of liver alcohol dehydrogenase, based on the X-ray diffraction structure for this enzyme. The addition of substrate and the associated conformation change induce an order—disorder transition for the solvent in the channel. A solvent network, connecting the active-site zinc ion and the protein surface, may provide the basis for a proton relay system. A molecular dynamics simulation of carbonic anhydrase showed two proton relay networks connecting the active-site zinc atom to the surrounding solvent (Vedani et ai, 1989). They remain intact when the substrate, HCOf, is bound. 

Aspartate aminotransferase catalyses the transfer of the NH2 group between aspartic acid and 2-oxoacids via the intervention of a pyridoxal coenzyme. The kinetically significant step, a 1,3-proton transfer, is thought to involve lysine-258 in a proton relay system (Scheme 18) replacement of lysine-258 with an alanine residue by site-directed muta-tion Q yields a mutant enzyme which is only weakly active but which can have activity restored by added amines (Scheme 18). 

The proposed catalytic mechanism involves a proton relay-type reaction where an immobilized HjO molecule serves as the nucleophile, a role normally filled by serine. The proton relay system is buried in the hydrophobic active-site wall. Ca " " binds the phosphate moiety of the substrate and, serving as a Lewis acid, polarizes the ester bond at the carbonyl oxygen. The HjO molecule, immobilized by the Asp-His pair attacks the carbonyl of the substrate and donates an H" " to His. The alkoxy oxygen of the glycerol backbone then retrieves the H" " from the His to complete the reaction. 

Fig. 9. The nicotinamide 2 -hydroxyl is implicated in a proton relay system for horse liver alcohol dehydrogenase (38). Thus electron density at the 2 -position induced during proton transfer can influence the microscopic redox potential by through-bond inductive effects.

The nature of the base that abstracts the proton on C2 in the various forms of enolase is not yet settled. For example, in the mechanism proposed on the basis of the crystal structure determination of the yeast enzyme, the base that is invoked in the forward reaction is the e-amino group of Lys345, while Glu211 interacts with the 3-hydroxyl group of substrate in the next stage of the reaction [74, 76]. In lobster enolase, on the other hand, the water molecule is part of the proton relay system that keeps the substrate in the carboxylic acid form this makes the pAT, of the C2 proton low enough for proton transfer to the base which, in this enzyme, may be His 157 [SO]. 

Tetrahedral Anions. The hydrolysis of the [BHJ anion in aqueous DMSO has the kinetic form for a general-acid-catalysed reaction, but with the catalytic coefficients and k-s.+ very different to the values observed for aqueous media. This difference is ascribed to the inability of DMSO to act as a proton relay system. A series of molecular orbital calculations suggest that BH5 could be a metastable intermediate in the hydrolysis of [BH They indicate the presence of identifiable BH3 and Hg subunits in the structure. The authors conclude, however, that limitations in the theoretical methods used do not yet allow experimental and computational results to be reconciled (at least to within a few kcal). The acid-cat ysed ammonolysis in liquid ammonia is first-order in both [NHJ+ and [BHJ, but is appreciably slower than the corresponding reaction in water the rate is markedly decreased by increases in ionic strength. This may be due to the participation of a water molecule in the latter case. The rates of reduction of a variety of substrates by [BHJ have been reported, and for the reduction of ketones it is suggested on the basis of H/T isotope effects that a four-centre transition state is involved. 

This amino acid contains the heterocyclic imidazole ring and possesses a unique chemistry. It is both a weak acid and weak base as well as an excellent nucleophile, and the only amino acid that has a pK which approximates physiological pH (7.35). As such, it can both pick up and dissociate protons within the biological milieu. Further, it may so function simultaneously by picking up a proton on one side of the ring, and donating it to the other. It has the potential of acting as a proton-relay system (detailed in Section 4.4.1). 

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