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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 9.2Making a Fast Reaction Faster: Carbonic Anhydrases

Carbon dioxide is a major end product of aerobic metabolism. In complex organisms, this carbon dioxide is released into the blood and transported to the lungs for exhalation. While in the blood, carbon dioxide reacts with water. The product of this reaction is a moderately strong acid, carbonic acid (pKa = 3.5), which becomes bicarbonate ion on the loss of a proton.

Image ch9fu4.jpg

Even in the absence of a catalyst, this hydration reaction proceeds at a moderate pace. At 37°C near neutral pH, the second-order rate constant k1 is 0.0027 M-1 s-1. This corresponds to an effective first-order rate constant of 0.15 s-1 in water ([H2O] = 55.5 M). Similarly, the reverse reaction, the dehydration of bicarbonate, is relatively rapid, with a rate constant of k-1 = 50 s-1. These rate constants correspond to an equilibrium constant of K1 = 5.4 × 10-5 and a ratio of [CO2] to [H2CO3] of 340:1.

Image caduceus.jpg Despite the fact that CO2 hydration and HCO3-dehydration occur spontaneously at reasonable rates in the absence of catalysts, almost all organisms contain enzymes, referred to as carbonic anhydrases, that catalyze these processes. Such enzymes are required because CO2 hydration and HCO3- dehydration are often coupled to rapid processes, particularly transport processes. For example, HCO3- in the blood must be dehydrated to form CO2 for exhalation as the blood passes through the lungs. Conversely, CO2 must be converted into HCO3- for the generation of the aqueous humor of the eye and other secretions. Furthermore, both CO2 and HCO3- are substrates and products for a variety of enzymes, and the rapid interconversion of these species may be necessary to ensure appropriate substrate levels. So important are these enzymes in human beings that mutations in some carbonic anhydrases have been found to cause osteopetrosis (excessive formation of dense bones accompanied by anemia) and mental retardation.

Carbonic anhydrases accelerate CO2 hydration dramatically. The most active enzymes, typified by human carbonic anhydrase II, hydrate CO2 at rates as high as kcat = 106 s-1, 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.

9.2.1. Carbonic Anhydrase Contains a Bound Zinc Ion Essential for Catalytic Activity

Less than 10 years after the discovery of carbonic anhydrase in 1932, this enzyme was found to contain bound zinc, associated with catalytic activity. This discovery, remarkable at the time, made carbonic anhydrase the first known zinc-containing enzyme. At present, hundreds of enzymes are known to contain zinc. In fact, more than one-third of all enzymes either contain bound metal ions or require the addition of such ions for activity. The chemical reactivity of metal ions—associated with their positive charges, with their ability to form relatively strong yet kinetically labile bonds, and, in some cases, with their capacity to be stable in more than one oxidation state—explains why catalytic strategies that employ metal ions have been adopted throughout evolution.

The results of x-ray crystallographic studies have supplied the most detailed and direct information about the zinc site in carbonic anhydrase. At least seven carbonic anhydrases, each with its own gene, are present in human beings. They are all clearly homologous, as revealed by substantial levels of sequence identity. Carbonic anhydrase II, present in relatively high concentrations in red blood cells, has been the most extensively studied (Figure 9.22).

Figure 9.22. The Structure of Human Carbonic Anhydrase II and Its Zinc Site.

Figure 9.22

The Structure of Human Carbonic Anhydrase II and Its Zinc Site. Image mouse.jpg (Left) The zinc is bound to the imidazole rings of three histidine residues as well as to a water molecule. (Right) The location of the zinc site in the enzyme.

Zinc is found only in the + 2 state in biological systems; so we need consider only this oxidation level as we examine the mechanism of carbonic anhydrase. A zinc atom is essentially always bound to four or more ligands; in carbonic anhydrase, three coordination sites are occupied by the imidazole rings of three histidine residues and an additional coordination site is occupied by a water molecule (or hydroxide ion, depending on pH). Because all of the molecules occupying the coordination sites are neutral, the overall charge on the Zn(His)3 unit remains +2.

9.2.2. Catalysis Entails Zinc Activation of Water

How does this zinc complex facilitate carbon dioxide hydration? A major clue comes from the pH profile of enzymatically catalyzed carbon dioxide hydration (Figure 9.23). At pH 8, the reaction proceeds near its maximal rate. As the pH decreases, the rate of the reaction drops. The midpoint of this transition is near pH 7, suggesting that a group with pKa = 7 plays an important role in the activity of carbonic anhydrase and that the deprotonated (high pH) form of this group participates more effectively in catalysis. Although some amino acids, notably histidine, have pKa values near 7, a variety of evidence suggests that the group responsible for this transition is not an amino acid but is the zinc-bound water molecule. Thus, the binding of a water molecule to the positively charged zinc center reduces the pKa of the water molecule from 15.7 to 7 (Figure 9.24). With the lowered pKa, a substantial concentration of hydroxide ion (bound to zinc) is generated at neutral pH. A zinc-bound hydroxide ion is sufficiently nucleophilic to attack carbon dioxide much more readily than water does. The importance of the zinc-bound hydroxide ion suggests a simple mechanism for carbon dioxide hydration (Figure 9.25).

Figure 9.23. Effect of pH on Carbonic Anhydrase Activity.

Figure 9.23

Effect of pH on Carbonic Anhydrase Activity. Changes in pH alter the rate of carbon dioxide hydration catalyzed by carbonic anhydrase II. The enzyme is maximally active at high pH.

Figure 9.24. The PKA of Water-Bound Zinc.

Figure 9.24

The PKA of Water-Bound Zinc. Binding to zinc lowers the pKa of water from 15.7 to 7.

Figure 9.25. Mechanism of Carbonic Anhydrase.

Figure 9.25

Mechanism of Carbonic Anhydrase. The zinc-bound hydroxide mechanism for the hydration of carbon dioxide catalyzed by carbonic anhydrase.

1.

Zinc facilitates the release of a proton from a water molecule, which generates a hydroxide ion.

2.

The carbon dioxide substrate binds to the enzyme's active site and is positioned to react with the hydroxide ion.

3.

The hydroxide ion attacks the carbon dioxide, converting it into bicarbonate ion.

4.

The catalytic site is regenerated with the release of the bicarbonate ion and the binding of another molecule of water.

Thus, the binding of water to zinc favors the formation of the transition state, leading to bicarbonate formation by facilitating proton release and by bringing the two reactants into close proximity. A range of studies supports this mechanism. In particular, studies of a synthetic analog model system provide evidence for its plausibility. A simple synthetic ligand binds zinc through four nitrogen atoms (compared with three histidine nitrogen atoms in the enzyme), as shown in Figure 9.26. One water molecule remains bound to the zinc ion in the complex. Direct measurements reveal that this water molecule has a pKa value of 8.7, not as low as the value for the water molecule in carbonic anhydrase but substantially lower than the value for free water. At pH 9.2, this complex accelerates the hydration of carbon dioxide more than 100-fold. Although catalysis by this synthetic system is much less efficient than catalysis by carbonic anhydrase, the model system strongly suggests that the zinc-bound hydroxide mechanism is likely to be correct. Carbonic anhydrases have evolved to utilize the reactivity intrinsic to a zinc-bound hydroxide ion as a potent catalyst.

Figure 9.26. A Synthetic Analog Model System for Carbonic Anhydrase.

Figure 9.26

A Synthetic Analog Model System for Carbonic Anhydrase. (A) An organic compound, capable of binding zinc, was synthesized as a model for carbonic anhydrase. The zinc complex of this ligand accelerates the hydration of carbon dioxide more than 100-fold (more...)

9.2.3. A Proton Shuttle Facilitates Rapid Regeneration of the Active Form of the Enzyme

As noted earlier, some carbonic anhydrases can hydrate carbon dioxide at rates as high as a million times a second (106 s-1). The magnitude of this rate can be understood from the following observations. At the conclusion of a carbon dioxide hydration reaction, the zinc-bound water molecule must lose a proton to regenerate the active form of the enzyme (Figure 9.27). The rate of the reverse reaction, the protonation of the zinc-bound hydroxide ion, is limited by the rate of proton diffusion. Protons diffuse very rapidly with second-order rate constants near 10-11 M-1 s-1. Thus, the backward rate constant k-1 must be less than 1011 M-1 s-1. Because the equilibrium constant K is equal to k1/k-1, the forward rate constant is given by k1 = K · k-1. Thus, if k-1 ≤ 1011 M-1 s-1 and K = 10-7 M (because pKa = 7), then k1 must be less than or equal to 104 s-1. In other words, the rate of proton diffusion limits the rate of proton release to less than 104 s-1 for a group with pKa= 7. However, if carbon dioxide is hydrated at a rate of 106 s-1, then every step in the mechanism (see Figure 9.25) must take place at least this fast. How can this apparent paradox be resolved?

Figure 9.27. Kinetics of Water Deprotonation.

Figure 9.27

Kinetics of Water Deprotonation. The kinetics of deprotonation and protonation of the zinc-bound water molecule in carbonic anhydrase.

The answer became clear with the realization that the highest rates of carbon dioxide hydration require the presence of buffer, suggesting that the buffer components participate in the reaction. The buffer can bind or release protons. The advantage is that, whereas the concentrations of protons and hydroxide ions are limited to 10-7 M at neutral pH, the concentration of buffer components can be much higher, on the order of several millimolar. If the buffer component BH+ has a pKa of 7 (matching that for the zinc-bound water), then the equilibrium constant for the reaction in Figure 9.28 is 1. The rate of proton abstraction is given by k1′ · [B]. The second-order rate constants k1′ and k-1′ will be limited by buffer diffusion to values less than approximately 109 M-1 s-1. Thus, buffer concentrations greater than [B] = 10-3 M (1 mM) may be high enough to support carbon dioxide hydration rates of 106 M-1 s-1 because k1′ · [B] = (109 M-1 s-1) · (10-3 M) = 106 s-1. This prediction is confirmed experimentally (Figure 9.29).

Figure 9.28. The Effect of Buffer on Deprotonation.

Figure 9.28

The Effect of Buffer on Deprotonation. The deprotonation of the zinc-bound water molecule in carbonic anhydrase is aided by buffer component B.

Figure 9.29. The Effect of Buffer Concentration on the Rate of Carbon Dioxide Hydration.

Figure 9.29

The Effect of Buffer Concentration on the Rate of Carbon Dioxide Hydration. The rate of carbon dioxide hydration increases with the concentration of the buffer 1,2-dimethylbenzimidazole. The buffer enables the enzyme to achieve its high catalytic rates. (more...)

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.

Figure 9.30. Histidine Proton Shuttle.

Figure 9.30

Histidine Proton Shuttle. (1) Histidine 64 abstracts a proton from the zinc bound water molecule, generating a nucleophilic hydroxide ion and a protonated histidine. (2) The buffer (B) removes a proton from the histidine, regenerating the unprotonated (more...)

9.2.4. Convergent Evolution Has Generated Zinc-Based Active Sites in Different Carbonic Anhydrases

Image tree.jpg Carbonic anhydrases homologous to the human enzymes, referred to as α-carbonic anhydrases, are common in animals and in some bacteria and algae. In addition, two other families of carbonic anhydrases have been discovered. The β-carbonic anhydrases are found in higher plants and in many bacterial species, including E. coli. These proteins contain the zinc required for catalytic activity but are not significantly similar in sequence to the α-carbonic anhydrases. Furthermore, the β-carbonic anhydrases have only one conserved histidine residue, whereas the α- carbonic anhydrases have three. No three-dimensional structure is yet available, but spectroscopic studies suggest that the zinc is bound by one histidine residue, two cysteine residues (conserved among β-carbonic anhydrases), and a water molecule. A third family, the γ-carbonic anhydrases, also has been identified, initially in the archaeon Methanosarcina thermophila. The crystal structure of this enzyme reveals three zinc sites extremely similar to those in the α-carbonic anhydrases. In this case, however, the three zinc sites lie at the interfaces between the three subunits of a trimeric enzyme (Figure 9.31). The very striking left-handed β-helix (a β strand twisted into a left-handed helix) structure present in this enzyme has also been found in enzymes that catalyze reactions unrelated to those of carbonic anhydrase. Thus, convergent evolution has generated carbonic anhydrases that rely on coordinated zinc ions at least three times. In each case, the catalytic activity appears to be associated with zinc-bound water molecules.

Figure 9.31. γ -Carbonic anhydrase.

Figure 9.31

γ -Carbonic anhydrase. Image mouse.jpg (Left) The zinc site of γ-carbonic anhydrase. (Middle) The trimeric structure of the protein (individual chains are labeled A, B, and C). (Right) The protein is rotated to show a top-down view that highlights its (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22599

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