1. Introduction: Overview of CA as a Model
Carbonic anhydrase (CA, EC 4.2.1.1) is a protein that is especially well-suited to serve as a model in many types of studies in biophysics, bioanalysis, the physical-organic chemistry of inhibitor design, and medicinal chemistry. In vivo, this enzyme catalyzes the hydration of CO2 and the dehydration of bicarbonate (eq 1).


CO2+H2O⇌HCO3−+H+

(1)


The active site of α-CAs comprises a catalytic ZnII ion coordinated by three imidazole groups of histidines and by one hydroxide ion (or water molecule), all in a distorted tetrahedral geometry. This grouping is located at the base of a cone-shaped amphiphilic depression, one wall of which is dominated by hydrophobic residues and the other of which is dominated by hydrophilic residues.1 Unless otherwise stated, “CA” in this review refers to (i) various isozymes of α-CAs or (ii) the specific α-CAs human carbonic anhydrases I and II (HCA I and HCA II) and bovine carbonic anhydrase II (BCA II); “HCA” refers to HCA I and HCA II; and “CA II” refers to HCA II and BCA II.

CA is particularly attractive for biophysical studies of protein–ligand binding for many reasons. (i) CA is a monomeric, single-chain protein of intermediate molecular weight (~30 kDa), and it has no pendant sugar or phosphate groups and no disulfide bonds. (ii) It is inexpensive and widely available. (iii) It is relatively easy to handle and purify, due in large part to its excellent stability under standard laboratory conditions. (iv) Amino acid sequences are available for most of its known isozymes. (v) The structure of CA, and of its active site, has been defined in detail by X-ray diffraction, and the mechanism of its catalytic activity is well-understood. (vi) As an enzyme, CA behaves not only as a hydratase/anhydrase with a high turnover number but also as an esterase (a reaction that is easy to follow experimentally). (vii) The mechanism of inhibition of CA by ligands that bind to the ZnII ion is fairly simple and well-characterized; it is, therefore, easy to screen inhibitors and to examine designed inhibitors that test theories of protein–ligand interactions. (viii) It is possible to prepare and study the metal-free apoenzyme and the numerous variants of CA in which the ZnII ion is replaced by other divalent ions. (ix) Charge ladders of CA II—sets of derivatives in which acylation of lysine amino groups (−NH3+ → −NHAc) changes the net charge of the protein—allow the influence of charge on properties to be examined by capillary electrophoresis. Some disadvantages of using CA include the following: (i) the presence of the ZnII cofactor, which can complicate biophysical and physical-organic analyses; (ii) a structure that is more stable than a representative globular protein and, thus, slightly suspect as a model system for certain studies of stability; (iii) a function—interconversion of carbon dioxide and carbonate—that does not involve the types of enzyme/substrate interactions that are most interesting in design of drugs; (iv) a catalytic reaction that is, in a sense, too simple (determining the mechanism of a reaction is, in practice, usually made easier if the reactants and products have an intermediate level of complexity); and (v) the absence of a solution structure of CA (by NMR spectroscopy). The ample X-ray data, however, paint an excellent picture of the changes (which are generally small) in the structure of CA that occur on binding ligands or introducing mutations.

The most important class of inhibitors of CA, the aryl-sulfonamides, has several characteristics that also make it particularly suitable for physical-organic studies of inhibitor binding and in drug design: (i) arylsulfonamides are easily synthesized; (ii) they bind with high affinity to CA (1 μM to sub-nM); (iii) they share one common structural feature; and (iv) they share a common, narrowly defined geometry of binding that exposes a part of the ligand that can be easily modified synthetically. There are also many non-sulfonamide, organic inhibitors of CA, as well as anionic, inorganic inhibitors.

We divide this review into five parts, all with the goal of using CA as a model system for biophysical studies: (I) an overview of the enzymatic activity and medical relevance of CA; (II) the structure and structure–function relationships of CA and its engineered mutants; (III) the thermodynamics and kinetics of the binding of ligands to CA; (IV) the effect of electrostatics on the binding of ligands to and the denaturation of CA; and (V) what makes CA a good model for studying protein–ligand binding and protein stability.



1.1. Value of Models 
CA serves as a good model system for the study of enzymes. That is, it is a protein having some characteristics representative of enzymes as a class, but with other characteristics that make it especially easy to study. It is a moderately important target in current medicinal chemistry: its inhibition is important in the treatment of glaucoma, altitude sickness, and obesity; its overexpression has recently been implicated in tumor growth; and its inhibition in pathogenic organisms might lead to further interesting drugs.2,3 More than its medical relevance, its tractability and simplicity are what make CA a particularly attractive model enzyme.

The importance of models in science is often underestimated. Models represent more complex classes of related systems and contribute to the study of those classes by focusing research on particular, tractable problems. The development of useful, widely accepted models is a critical function of scientific research: many of the techniques (both experimental and analytical) and concepts of science are developed in terms of models; they are thoroughly engrained in our system of research and analysis.

Examples of models abound in successful areas of science: in biology, E. coli, S. cerevisiae, Drosophila mela-nogaster, C. elegans, Brachydanio rerio (zebrafish), and the mouse; in chemistry, the hydrogen atom, octanol as a hydrophobic medium, benzene as an aromatic molecule, the 2-norbornyl carbocation as a nonclassical ion, substituted cyclohexanes for the study of steric effects, p-substituted benzoic acids for the study of electronic effects, cyclodextrins for ligand–receptor interactions; in physics, a vibrating string as an oscillator and a particle in a box as a model for electrons in orbitals.

Science needs models for many reasons:


Focus: Models allow a community of researchers to study a common subject. Solving any significant problem in science requires a substantial effort, with contributions from many individuals and techniques. Models are often the systems chosen to make this productive, cooperative focus possible.


Research Overhead: Development of a system to the point where many details are scientifically tractable is the product of a range of contributions: for enzymes, these contributions are protocols for preparations, development of assays, determination of structures, preparation of mutants, definition of substrate specificity, study of rates, and development of mechanistic models. In a well-developed model system, the accumulation of this information makes it relatively easy to carry out research, since before new experiments begin, much of the background work–the fundamental research in a new system–has already been carried out.


Recruiting and Interdisciplinarity: The availability of good model systems makes it relatively easy for a neophyte to enter an area of research and to test ideas efficiently. This ease of entry recruits new research groups, who use, augment, and improve the model system. It is especially important to have model systems to encourage participation by researchers in other disciplines, for whom even the elementary technical procedures in a new field may appear daunting.


Comparability: A well-established model allows researchers in different laboratories to calibrate their experiments, by reproducing well-characterized experiments.


Community: The most important end result of a good model system is often the generation of a scientific community–that is, a group of researchers examining a common problem from different perspectives and pooling information relevant to common objectives.



One of the goals of this review is to summarize many experimental and theoretical studies of CA that have established it as a model protein. We hope that this summary will make it easier for others to use this protein to study fundamentals of two of the most important questions in current chemistry: (i) Why do a protein and ligand associate selectively? (ii) How can one design an inhibitor to bind to a protein selectively and tightly? We believe that the summary of studies of folding and stability of CA will be useful to biophysicists who study protein folding. In addition, we hope that the compilation of data relevant to CA in one review will ease the search for information for those who are beginning to work with this protein.

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Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein–Ligand Binding

Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream In contrast, the conventional antibodies apparently do not interact with the enzyme's active site Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes In addition we show that among those isolated fragments active site binders are well represented When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors

Potent enzyme inhibitors derived from dromedary heavy‐chain antibodies

Molecular recognition between proteins and their interacting partners underlies the biochemistry of living organisms. Specificity in this recognition is thought to be essential, whereas promiscuity is often associated with unwanted side effects, poor catalytic properties and errors in biological function. Recent experimental evidence suggests that promiscuity, not only in interactions but also in the actual function of proteins, is not as rare as was previously thought. This has implications not only for our fundamental understanding of molecular recognition and how protein function has evolved over time but also in the realm of biotechnology. Understanding protein promiscuity is becoming increasingly important not only to optimize protein engineering applications in areas as diverse as synthetic biology and metagenomics but also to lower attrition rates in drug discovery programs, identify drug interaction surfaces less susceptible to escape mutations and potentiate the power of polypharmacology.

Protein promiscuity and its implications for biotechnology

Monographs on fragrance raw materials

Summary

1. Carbonic anhydrases from vertebrates, plants and bacteria have molecular weights of 30,000 (or multiples thereof), contain one zinc atom per 30,000 molecular weight, and are inhibited by acetazolamide and related compounds.



2. In mammals, there are two major isoenzymes of carbonic anhydrase. The so-called ‘high activity’ carbonic anhydrase possesses a carbon dioxide-hydratase activity many times (in the case of the guinea-pig isoenzymes, 18 times) that of the ‘low activity’ isoenzyme.



3. The mammalian isoenzymes differ from one another in their amino-acid compositions (the difference in serine contents being a consistent finding in a number of species), in their physical properties (isoelectric pH, retention by ion-exchange resins, electrophoretic mobility) and in their kinetic properties.



4. Mammalian carbonic anhydrases also catalyse the hydrolysis of some esters and the hydration of aldehydes. Their relative activities with these other substrates may be substantially different from their relative activities with carbon dioxide as substrate. The discovery that carbonic anhydrase may also catalyse other reactions raises the possibility that the enzyme may have other roles in metabolic pathways (e.g., in certain dehydrogenation reactions).



5. In plants, the role of carbonic anhydrase may be to catalyse the inter-conversion of bicarbonate and carbon dioxide, to provide ‘carbon dioxide’ in the form appropriate for carbon-fixing reactions. A similar role has been suggested for the carbonic anhydrase found in Neissariae.



6. The carbonic anhydrase of vertebrates (the ‘high activity’ isoenzyme of mammals) is found in many ion-transporting epithelia, but its role in them is still uncertain. It occurs in acid-transporting epithelia such as the stomach and kidney, and also in certain bicarbonate-transporting epithelia like the large intestine. There is, however, a poor correlation between the presence of the enzyme and the occurrence of acid or bicarbonate secretion. Thus there is little or no carbonic anhydrase in some tissues noted for their ability to secrete bicarbonate ions, such as the pancreas and ileum. Conversely, carbonic anhydrase-containing tissues like the avian salt gland and the elasmobranch rectal gland form concentrated sodium chloride solutions which are of nearly neutral pH. The correlation between the distribution of carbonic anhydrase and the occurrence of active chloride transport appears similar to that between carbonic anhydrase and bicarbonate ion transport.



7. While the red cells of most mammalian species so far studied contain both ‘high activity’ and ‘low activity’ isoenzymes, the latter is reported to be absent from the erythrocytes of sheep, ox and dog. Presumably the ‘low activity’ isoenzyme is not necessary for an adequate rate of carbon dioxide exchange to occur between tissues and lungs in these species.



8. The ‘low activity’ isoenzyme is present in tissues other than blood, for example, the colon, caecum and ox rumen, and probably also, the gall bladder and kidney medulla. Its distribution differs markedly from that of the ‘high activity’ isoenzyme and presumably it has a particular functional importance of its own. It is not possible to define the role of the ‘low activity’ isoenzyme at present, but attention is drawn to the possibility that it is concerned in the handling of the products of microbial fermentation, such as ammonia and organic acids, by the large intestines and ruminant forestomach.



My work on the isoenzymes of carbonic anhydrase was supported by a Medical Research Council Scholarship, and by an equipment grant to Dr D. S. Parsons. I am also grateful to Dr D. S. Parsons for his comments on the manuscript.

Y Pocker

Papers

The catalytic versatility of erythrocyte carbonic anhydrase. VII. Kinetic studies of esterase activity and competitive inhibition by substrate analogs