Carbonic anhydrase proton transfer rate

As an example, consider an early calculation of isotope effects on enzyme kinetics by Hwang and Warshel [31]. This study examines isotope effects on the catalytic reaction of carbonic anhydrase. The expected rate-limiting step is a proton transfer reaction from a zinc-bound water molecule to a neighboring water. The TST expression for the rate constant k is... 

As an illustration, we briefly discuss the SCC-DFTB/MM simulations of carbonic anhydrase II (CAII), which is a zinc-enzyme that catalyzes the interconversion of CO2 and HCO [86], The rate-limiting step of the catalytic cycle is a proton transfer between a zinc-bound water/hydroxide and the neutral/protonated His64 residue close to the protein/solvent interface. Since this proton transfer spans at least 8-10 A depending on the orientation of the His 64 sidechain ( in vs. out , both observed in the X-ray study [87]), the transfer is believed to be mediated by the water molecules in the active site (see Figure 7-1). To carry out meaningful simulations for the proton transfer in CAII, therefore, it is crucial to be able to describe the water structure in the active site and the sidechain flexibility of His 64 in a satisfactory manner. 

Carbonic anhydrase presents an instructive case where the catalytic efficiency is so great (kcat > 10 s- ) that proton transfer becomes rate-limiting. The rate was found to depend on the concentration of the protonated form of buffers in the solution. Indeed, Silverman and Tu adduced the first convincing evidence for the role of buffer in carbonic anhydrase catalysis through their observation of an imidazole buffer-dependent enhancement in equilibrium exchanges of oxygen isotope between carbon dioxide and water. The effect is strictly on kcat, and is unaffected because the latter is... 

Importantly, carbonic anhydrase II is one of the most efficient biological catalysts known and it catalyzes the hydration of CO2 with a turnover rate of 10 sec at 25 C (Khalifah, 1971 Steiner et al, 1975). With kcaJKm = 1.5 X 10 sec carbonic anhydrase II is one of a handful of enzymes for which catalysis apparently approaches the limit of diffusion control. Since transfer of the product proton away from the enzyme to bulk solvent comprises a kinetic obstacle [an enzyme-bound group with ap/C, of about 7 cannot transfer a proton to bulk solvent at a rate faster than 10 sec (for a review see Eigen and Hammes, 1963)], the observed turnover rate of 10 sec" requires the participation of buffer in the proton transfer. 

The importance of maintaining the active site water network in CA II for efficient proton transfer was investigated by substituting different amino acids of varying size at position 65 and measuring the rate constants for proton transfer in the variant carbonic anhydrases... 

It was once thought that the rate of equilibrium of the catalytic acid and basic groups on an enzyme with the solvent limited the rates of acid- and base-catalyzed reactions to turnover numbers of 103 s 1 or less. This is because the rate constants for the transfer of a proton from the imidazolium ion to water and from water to imidazole are about 2 X 103 s 1. However, protons are transferred between imidazole or imidazolium ion and buffer species in solution with rate constants that are many times higher than this. For example, the rate constants with ATP, which has a pKa similar to imidazole s, are about I0 J s 1 M-1, and the ATP concentration is about 2 mM in the cell. Similarly, several other metabolites that are present at millimolar concentrations have acidic and basic groups that allow catalytic groups on an enzyme to equilibrate with the solvent at 107 to 108 s-1 or faster. Enzyme turnover numbers are usually considerably lower than this, in the range of 10 to 103 s-1, although carbonic anhydrase and catalase have turnover numbers of 106 and 4 X 107 s 1, respectively. 

The calculated [using a quantized classical path (QCP) approach] and observed isotope effects and rate constants are in good agreement for the proton-transfer step in the catalytic reaction of carbonic anhydrase. This approach takes account of the role of quantum mechanical nuclear motions in enzyme reactions.208... 

In fact, transient assembly of H-bonded water files is probably common in enzyme function. In carbonic anhydrase, for example, the rate-limiting step is proton transfer from the active-site Zn2+-OH2 complex to the surface, via a transient, H-bonded water network that conducts H+. Analysis of the relationship between rates and free energies (p K differences) by standard Marcus theory shows that the major contribution to the observed activation barrier is in the work term for assembling the water chain (Ren et al., 1995). 

Carbonic anhydrases accelerate CO2 hydration dramatically. The most active enzymes, typified by human carbonic anhydrase II, hydrate CO2 at rates as high as k =10 s, or a million times a second. Fundamental physical processes such as diffusion and proton transfer ordinarily limit the rate of hydration, and so special strategies are required to attain such prodigious rates. 

Carbonic anhydrases catalyze the reaction of water with carbon dioxide to generate carbonic acid. The catalysis can be extremely fast molecules of some carbonic anhydrases hydrate carbon dioxide at rates as high as 1 million times per second. A tightly bound zinc ion is a crucial component of the active sites of these enzymes. Each zinc ion binds a water molecule and promotes its deprotonation to generate a hydroxide ion at neutral pH. This hydroxide attacks carbon dioxide to form bicarbonate ion, HCO3 ". Because of the physiological roles of carbon dioxide and bicarbonate ions, speed is of the essence for this enzyme. To overcome limitations imposed by the rate of proton transfer from the zinc-bound water molecule, the most active carbonic anhydrases have evolved a proton shuttle to transfer protons to a buffer. 

Silverman D. N. (1982) Carbonic-anhydrase—exchange catalyzed by an enzyme with rate contributing proton transfer steps. Meth. Enzymol. 87, 732-752. 

Silverman, D.N., et al. (1993). Rate-equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III. Biochemistry 34, 10757-10762... 

A small isotope effect for proton transport through a water chain is also observed for catalytic conversion of CO2 to HCO by carbonic anhydrase II [68, 69]. The rate-determining step in this process is the transfer of a proton from the H2O ligand of a four-coordinated zinc ion to a histidine residue located at a distance of about 8 A. The proton conduit is known to consists of water molecules located in a pocket that can contain several such molecules, which are freely exchanged with... 

Figure 29.19 The reactant (R), transition state (TS) and product (P) configurations for the rate-determining triple proton transfer step of the 58-atom model used to represent the active site of carbonic anhydrase II [18]. The numbers denote bond distances (in A) calculated at two different levels of theory. The arrows in the insert figure represent the tunneling mode and illustrate the degree of synchronicity of the transfer.

Silverman, D. N., Tu, C., Chen, X., Tanhauser, S. M., Kresge, A. J., Iaipis, P. j., Rate-Equilibria Relationships in Intramolecular Proton Transfer in Human Carbonic Anhydrase III, Biochemistry 1993, 34, 10757-10762. 

Although usually high, the proton-transfer steps may become the rate-limiting steps in catalysis. If the proton movement into and out of the active site is restricted, the state of protonation of the enzyme-substrate complex is not equilibrated rapidly with respect to the rate of reaction to give products or the rate of substrate dissociation (Section 14.6.2) that is why the enzymes have acid-base catalysis. On the other hand, when the chemical step is exceptionally fast, the proton-transfer reactions may become the rate-limiting steps in catalysis this case is verified in reaction catalyzed by carbonic anhydrase (Silverman Tu, 1975). 

Carbonic anhydrase is one of the rare enzymes in which the chemical step of catalysis is so fast that intramolecular proton transfer is rate limiting, which allows this transfer to be studied in detail. 

Stopped-flow methods have also been used to study the rapid reactions of CO2 with [Co(NH8)8(OH)] + ion in H2O and D2O. The solvent isotope effect is 1.0 as expected for a reaction not involving a rate-determining proton transfer. However, an isotope effect of ca. 3 has been reported for the bovine carbonic anhydrase-catalysed hydration of CO2. It is argued that if the zinc-hydroxo-complex in the enzyme is acting as a nucleophile in the hydration of CO2, then an isotope effect of unity would be expected. On the other hand, if the zinc-hydroxo-complex, or any other amino-... 

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