Proton transfers, carbonic anhydrase

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. 

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... 

The identification of different carbonate binding modes in copper(II) and in zinc(II)/2,2 -bipyridine or tris(2-aminoethyl)amine/(bi)carbonate systems, specifically the characterization by X-ray diffraction techniques of both r)1 and r 2 isomers of [Cu(phen)2(HC03)]+ in their respective perchlorate salts, supports theories of the mechanism of action of carbonic anhydrase which invoke intramolecular proton transfer and thus participation by r)1 and by r 2 bicarbonate (55,318). 

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. 

There may be two proton transfers in the carbonic anhydrase II-catalyzed mechanism of CO2 hydration that are important in catalysis, and both of these transfers are affected by the active-site zinc ion. The first (intramolecular) proton transfer may actually be a tautomerization between the intermediate and product forms of the bicarbonate anion (Fig. 28). This is believed to be a necessary step in the carbonic anhydrase II mechanism, due to a consideration of the reverse reaction. The cou-lombic attraction between bicarbonate and zinc is optimal when both oxygens of the delocalized anion face zinc, that is, when the bicarbonate anion is oriented with syn stereochemistry toward zinc (this is analogous to a syn-oriented carboxylate-zinc interaction see Fig. 28a). This energetically favorable interaction probably dominates the initial recognition of bicarbonate, but the tautomerization of zinc-bound bicarbonate is subsequently required for turnover in the reverse reaction (Fig. 28b). 

Experimental evidence in support of bicarbonate tautomerization has been presented by Pocker and Deits (1983), who reported that alkyl carbonates ROCO2 bind to carbonic anhydrase II, but are not active in the reverse reaction. If only the reorientation of the zinc-bound carbonate were required for catalytic activity, it would not be unreasonable to expect carbonic anhydrase II to catalyze the degradation of alkyl carbonates. However, alkyl carbonates cannot undergo the proton transfer... 

Another contrast between the zinc proteases and the carbonic an-hydrases concerns the zinc coordination polyhedron. The carbonic an-hydrases ligate zinc via three histidine residues, whereas the zinc proteases ligate the metal ion through two histidine residues and a glutamate (bidentate in carboxypeptidase A, unidentate in thermolysin). Hence, the fourth ligand on each catalytic zinc ion, a solvent molecule, experiences enhanced electrostatic polarization in carbonic anhydrase II relative to carboxypeptidase A. Indeed, the zinc-bound solvent of carbonic anhydrase II is actually the hydroxide anion [via a proton transfer step mediated by His-64 (for a review see Silverman and Lindskog, 1988)]. 

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... 

CARBONATO COMPLEXES MODELS FOR CARBONIC ANHYDRASE Lipscomb Pathway (Proton Transfer)... 

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... 

General acid-base catalysis is often the controlling factor in many mechanisms and acts via highly efficient and sometimes intricate proton transfers. Whereas log K versus pH profiles for conventional acid-base catalyzed chemical processes pass through a minimum around pH 7.0, this pH value for enzyme reactions is often the maximum. In enzymes, the transition metal ion Zn2+ usually displays the classic role of a Lewis acid, however, metal-free examples such as lysozyme are known too. Good examples of acid-base catalysis are the mechanisms of carbonic anhydrase II and both heme- and vanadium-containing haloperoxidase. 

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. 

The molecular components of many buffers are too large to reach the active site of carbonic anhydrase. Carbonic anhydrase II has evolved a proton shuttle to allow buffer components to participate in the reaction from solution. The primary component of this shuttle is histidine 64. This residue transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer (Figure 9.30). Thus, catalytic function has been enhanced through the evolution of an apparatus for controlling proton transfer from and to the active site. Because protons participate in many biochemical reactions, the manipulation of the proton inventory within active sites is crucial to the function of many enzymes and explains the prominence of acid-base catalysis. 

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. 

Figure ]4r Calculated free energy profiles for the reference reaction, 21 0 OH l HjO+ (open triangles) in water and for the proton transfer step in human carbonic anhydrase 1 (open squares). The calculated energetics of proton transfer from a zinc-bound water molecule in aqueous solution is also shown for comparison (Aqvist...

Warshel, A., Hwang, J.K. and Aqvist, J. (1992). Computer simulations of enzymatic reactions examination of linear free-energy relationships and quantum-mechanical corrections in the initial proton-transfer step of carbonic anhydrase. Faraday Discuss 93, 225... 

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

---