Structural annotation of human carbonic anhydrases

Abstract

Carbonic anhydrases (CAs, EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible interconversion of CO₂ and HCO₃⁻. Of the 15 isoforms of human (h) α-CA, 12 are catalytic (hCAs I-IV, VA, VB, VI, VII, IX, XII-XIV). The remaining three acatalytic isoforms (hCAs VIII, X and XI) lack the active site Zn²⁺ and are referred to as CA-related proteins (CA-RPs); however, their function remains elusive. Overall these isoforms are very similar to each other in structure but they differ in their expression and distribution. The favourable properties of hCA II such as fast kinetics, easy expression and purification, high solubility and intermediate heat resistance have made it an attractive candidate for numerous industrial applications. This review highlights the structural similarity and stability comparison among hCAs.

Keywords::

• Human carbonic anhydrase
• annotation
• classification
• comparison
• thermostability
• industrial applications

Classification

Carbonic anhydrases (CAs, EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate ion^1,2. CAs are found in both prokaryotes and eukaryotes, and are involved in many physiological processes such as respiration, bone resorption, calcification and photosynthesis³.

CAs have been divided into five distinct classes (α, β, γ, δ and ζ). The α-class is found in mammals, β is found in plants and some prokaryotes, γ is present only in archaeabacteria and two other rare classes (that are similar to class β), δ and ζ are found in diatoms^4–9. The three main classes (α, β and γ) of CA are structurally dissimilar and are thought to have evolved independently, possibly as a result of convergent evolution.

Structure and function of human CAs

Structure

There are 15 isoforms of human (h) α-CA of which, three are acatalytic and will be discussed later. The 12 catalytic isoforms differ from each other in catalytic activity, tissue localization and distribution (Table 1) in forms of cytosolic (hCAs I-III, VII, XIII), membrane bound (hCAs IV, IX, XII, XIV), secretory (hCA VI) or mitochondrial (hCAs VA, VB)^7,10–25. Overall, most hCA isoforms (except hCA VI, IX and XII) exist in monomeric forms composed of seven right handed α-helices, and a twisted β-sheet formed by 10 β-strands (two parallel and eight antiparallel). The CA catalytic domains in transmembrane hCA IX and hCA XII have a similar, but dimeric structure. Figure 1 shows a primary sequence alignment of all the isoforms of hCA, and Tables 2 and 3 summarize the primary sequence identity and structural similarity, respectively. A superposition of all the catalytic isoforms of hCA shows high similarity in the main chain (Figure 2).

Table 1.  Catalytic efficiency and distribution of hCA isoforms.

Isoform	kcat (s^−1)	KM (mM)	kcat/KM (M^−1 s^−1)	Localization		Ref
				Sub-cellular	Tissue/Organ
hCA I	2.0 × 10^5	4.0	5.0 × 10^7	Cytosol	RBCs, GI tract	^Citation5,Citation9,Citation95
hCA II	1.4 × 10^6	9.3	1.5 × 10^8	Cytosol	RBCs, GI tract, eyes, Osteoclasts, kidneys, lungs, testes, brain	^Citation5,Citation9,Citation95
hCA III	1.0 × 10^4	33.3	3.0 × 10^5	Cytosol	Skeletal muscles, adipocytes	^Citation5,Citation9,Citation95
hCA IV	1.1 × 10^6	21.5	5.1 × 10^7	Membrane-bound	Kidneys, lungs, pancreas, brain, capillaries, colon, heart muscles	^Citation5,Citation9
hCA VA	2.9 × 10^5	10.0	2.9 × 10^7	Mitochondria	Liver	^Citation12
hCA VB	9.5 × 10^5	9.7	9.8 × 10^7	Mitochondria	Heart and skeletal muscles, pancreas, kidneys, GI tract, spinal cord	^Citation12
hCA VI	3.4 × 10^5	6.9	4.9 × 10^7	Secretory (milk/saliva)	Salivary and mammary glands	^Citation13
hCA VII	9.5 × 10^5	11.4	8.3 × 10^7	Cytosol	CNS	^Citation14
hCA-RP VIII	–			Cytosol	CNS	^Citation5,Citation9,Citation95
hCA IX	3.8 × 10^5	6.9	5.5 × 10^7	Transmembrane	Tumours, GI mucosa	^Citation95,Citation96
hCA-RP X	–			Cytosol	CNS	^Citation5,Citation9,Citation95
hCA-RP XI	–			Cytosol	CNS	^Citation5,Citation9,Citation95
hCA XII	4.2 × 10^5	12.0	3.5 × 10^7	Transmembrane	Renal, intestinal, reproductive epithelia, eye, tumours	^Citation89
hCA XIII	1.5 × 10^5	13.8	1.1 × 10^7	Cytosol	Kidneys, brain, lungs, gut, reproductive tract	^Citation18
hCA XIV	3.1 × 10^5	7.9	3.9 × 10^7	Transmembrane	Kidneys, brain, liver	^Citation90

Table 2.  Primary sequence identity (upper right) and number of conserved residues (lower left) among hCA isoforms.

	I	II	III	IV	V	VI	VII	IX	XII	XIV
I	-	60.5	54.4	28.8	43.7	30.9	50.6	31.3	29.8	32.6
II	158	-	58.9	32.4	49.4	32.5	55.9	31.4	28.4	36.0
III	142	153	-	30.2	43.3	32.8	52.9	29.5	27.3	33.0
IV	78	88	82	-	27.9	31.3	33.1	29.4	26.0	34.2
V*	114	129	113	76	-	28.6	47.9	29.0	24.3	32.6
VI	83	87	88	85	77	-	35.3	41.0	35.4	41.4
VII	132	146	138	90	125	95	-	34.6	31.6	36.3
IX	85	85	80	80	79	110	94	-	31.2	41.9
XII	79	75	72	70	64	93	84	83	-	37.8
XIV	86	82	88	93	87	110	97	113	99	-

*Murine CA.

Table 3.  Structural main chain r.m.s.d. (Å) among hCA isoforms.

	I	II	III	IV	V*	VI	VII	IX	XII	XIII	XIV
I	-	0.98	0.87	2.48	1.02	1.55	0.99	2.02	2.07	0.85	1.52
II		-	0.96	2.29	0.98	1.48	0.77	2.05	2.17	0.76	1.50
III			-	2.37	1.01	1.69	0.86	1.79	1.99	0.85	1.41
IV				-	1.65	1.52	2.36	2.81	2.78	1.39	2.48
V*					-	1.47	0.83	1.82	1.89	1.30	1.42
VI						-	1.52	1.35	1.58	1.58	1.49
VII							-	1.79	1.97	0.77	1.42
IX								-	1.87	2.06	1.88
XII									-	1.17	1.86
XIII										-	1.18
XIV*											-

*Murine CA.

Figure 1.  Multiple sequence alignment of the hCA isoforms. Gradients of blue represent sequence conservation from most (dark blue) to least conserved (light blue).

Figure 2.  (A) Cartoon representation of superposition of the 11 catalytic isoforms of α-CA, in shades of blue. The histidines (94, 96 and 119) that coordinate the Zn²⁺ are shown as yellow sticks. (B) Worm representation of main chain r.m.s.d. of the 11 catalytic isoforms. Thicker regions represent more deviation in the main chain. Active site Zn²⁺ is represented as a magenta sphere. Figure was made using Chimera¹¹⁰.

HCA II is the most well studied of all CA isoforms and its active site can be described as a cone-shaped cavity formed of a hydrophobic region (Val121, Val143, Leu198, Val207 and Trp209), and a hydrophilic region (Tyr7, Asn62, His64, Asn67, Thr199 and Thr200). At the base of the cavity lies the Zn²⁺, tetrahedrally coordinated by three conserved histidines (His94, His96 and His119) and a solvent molecule (Figure 3). Although the core of the active site in α-CAs is highly conserved, there is variability in the polarity and hydropathicity of its periphery. The conservation of polar and non-polar residues on the surface of catalytic isoforms of hCA is shown in Figures 4 and 5, respectively.

Figure 3.  Stick representation of the active site of hCA II (PDB ID 3D92)² showing key residues that are involved in CO₂ binding and proton transfer during catalysis. Residues are as labelled. Active site Zn²⁺ is shown as a magenta sphere and water molecules involved in proton transfer are shown as red spheres.

Figure 4.  Surface representation of conservation of hydrophobic amino acids among the catalytic α-CAs. The conserved residues are coloured in a gradient of dark (most conserved) to light (least conserved) green. Active site Zn²⁺ is shown as a magenta sphere. Figure was made using Chimera¹¹⁰.

Figure 5.  Surface representation of conservation of hydrophilic amino acids among the catalytic α-CAs. The conserved residues are coloured in a gradient of red (negatively charged) and blue (positively charged), from dark (most conserved) to light (least conserved). Active site Zn²⁺ is shown as a magenta sphere. Figure was made using Chimera¹¹⁰.

Catalytic mechanism

CAs follow a two-step ping pong catalytic mechanism for the hydration and dehydration of CO₂ and HCO₃⁻, respectively:

Step 1: EZnOH⁻ + CO₂ ↔ EZnHCO₃⁻ ↔ EZnH₂O + HCO₃⁻

Step 2: EZnH₂O + B ↔ EZnOH⁻ + BH⁺

The first step of the reaction involves the binding of a CO₂ molecule in the active site². Molecular dynamics (MD) data suggest the existence of three CO₂ binding sites within the active site, one of which is found in the hydrophobic region (3–4 Å away from Zn²⁺), and the other two are found 6–7 Å away from the Zn²⁺. Energy analysis suggests that the two binding sites that are 6–7 Å away may act as intermediates guiding CO₂ into the hydrophobic region²⁶. However, the recent X-ray crystal structure (PDB ID: 3D92) reveals a single binding site for CO₂ in the active site of hCA II with one of the oxygens of the CO₂ interacting with the amide of Thr199 (3.5 Å) and displacing a water molecule whereas, the other oxygen is positioned between the Zn²⁺ and Val121. This arrangement places both of the oxygens nearly equidistant from the zinc-bound OH⁻ (Zn-OH⁻), and the carbon 2.8 Å from the Zn-OH⁻². Once inside this hydrophobic region, the CO₂ undergoes a nucleophillic attack by the Zn-OH⁻, forming zinc-bound bicarbonate (Zn-HCO₃⁻). This HCO₃⁻ is then displaced with a water molecule (Zn-H₂O) and the former diffuses into the bulk solvent. In the second step, the Zn-OH⁻ is regenerated as a result of the transfer of a proton from the Zn-H₂O generated in step one, to the bulk solvent (or buffer)⁸.

Kinetic studies show differences in catalytic efficiency among hCAs (Table 1) which may be attributed to the varying speed of proton shuttling (the rate determining step of catalysis) in step two²⁶. The proton shuttling step is said to occur by two key events. The first event involves the transfer of the proton via a network of ordered water molecules in the active site to a nearby residue, while the second subsequently releases the proton into the bulk solvent²⁷.

One of the key amino acid residues in proton shuttling is His64 (hCA II numbering), which is conserved in all of the catalytic hCAs except III and V. In X-ray crystal structures²⁸ and MD studies²⁹, His64 has been found to exist in a dual conformation: inward (7.5 Å away from the Zn²⁺) and outward (12.0 Å away from the Zn²⁺) suggesting that the residue acts as a shuttle, accepting the proton then transferring it to the bulk solvent by a flipping mechanism. Although, there exists another hypothesis according to which it is not the flipping of the side chain of His64, but its tautomerism that results in proton transfer³⁰. Absence of His64 in hCAs III and V could well be argued as the reason for their relatively slowed catalytic efficiency. However, the rate of proton transfer also depends on the number of water molecules involved in proton transfer, as well as the distance between the proton donor and acceptor³¹.

In hCA III, mainly three residues are speculated to affect catalysis: Lys64, Arg67 and Phe198 in place of His64, Asn67 and Leu198 (hCA II numbering)³². The loss of His64 for a much more basic Lys, and presence of bulkier Arg67 and Phe198 causes a reduction in the active site cavity volume affecting the binding of CO₂, thereby resulting in a lower rate of catalysis³³. However, the exact mechanism of how the proton leaves the active site and the exact role of these residues is still unanswered. In CA V, presence of larger residues, Tyr and Phe, in place of His64 and Ala65 (CA II numbering) respectively, makes the region very hydrophobic and thus these residues are believed to be not involved in proton transfer. It is speculated however, that the proton travels through another network possibly involving either Tyr131 or Lys132³⁴.

Up until now, the primary reaction i.e. reversible hydration of carbon dioxide to bicarbonate ion has been discussed. However, CA has been known to be involved in a variety of other reactions (Table 4), mechanisms and physiological functions of which are yet to be ascertained³.

Table 4.  Reactions that CAs are believed to be involved in³.

O=C=O + H2O ↔ HCO3^− + H^+

O=C=NH + H2O ↔ H2NCOOH

HN=C=NH + H2O ↔ H2NCONH2

RCHO + H2O ↔ RCH(OH)2

RCOOAr + H2O ↔ RCOOH + ArOH

RSO3Ar + H2O ↔ RSO3H + ArOH

ArF + H2O ↔ HF + ArOH

Ar = 2,4 – dinitrophenyl

PhCH2OCOCl + H2O ↔ PhCH2OH + CO2 + HCl

RSO2Cl + H2O ↔ RSO3H + HCl

R = Methyl or Phenyl (Ph)

Physiological functions

The isoforms of hCA are found throughout the body in various tissues and sub-cellular locations and have varied physiological roles (Table 1). hCAs I, II and III share a high sequence homology, and have been extensively studied. hCAs I, II and IV are involved in the efficient transportation of CO_2. hCA II is the most widely expressed isoform and participates in processes ranging from bone resorption to respiration and pH regulation. hCA III is believed to serve an anti-oxidant role in cells which have high rates of oxidation such as adipose tissues, hepatocytes and skeletal muscle fibers³⁵.

hCA V is found in the mitochondrial matrix, existing in two forms: VA and VB. hCA VA provides bicarbonate for gluconeogenesis, and fatty acids for pyrimidine base synthesis³. It is found in the mitochondria of the heart, lung, kidney, spleen and intestines. hCA VB on the other hand, is present in the mitochondria of the pancreas, kidney and salivary glands playing an intermediate role in metabolism³⁶.

hCA VI is the secretory isoform expressed in saliva, milk, nasal secretions and the epithelial lining of digestive organs. The physiological function of hCA VI is yet to be fully understood, but studies show a correlation between the loss of taste and a decrease in hCA VI secretion³⁷. hCA VII (cytosolic) is speculated to play a role in cerebrospinal fluid production in the CNS³⁶. hCA XIII is another cytosolic isoform that is expressed among tissues in the reproductive organs and is speculated to play a role in pH regulation, and ensuring proper fertilization³⁸.

Among the three transmembrane hCAs, isoforms IX and XII are expressed in the gastrointestinal mucosa. However, they have also been found to be over expressed in epithelial tumours including tumours of the cervix, lungs, kidneys, prostate and breast. In addition, these isoforms are implicated in allowing tumours to acclimate to a hypoxic microenvironment and promoting metastasis³⁹. hCA XIV is present in the brain and retina and is believed to aid in the removal of CO₂ from the neural retina and help modulate photoreceptor function⁴⁰.

CA-related proteins

The hCA family, besides the catalytic isoforms, includes a subclass of three non-catalytic isoforms (hCAs VIII, X and XI) called CA-related proteins (CA-RPs), based on sequence homology with the catalytic isoforms. The reason behind these isoforms being non-catalytic has been attributed to the absence of one or more histidines that coordinate the Zn²⁺ ion in the active site of a catalytic hCA isoform. In hCA-RP VIII, for example, the Zn-coordinating His94 (hCA II numbering) is replaced by Arg116 (hCA-RP VIII numbering)⁴¹ which precludes CO₂ hydration in the first step of CA catalysis⁴². Although the biological functions of CA-RPs remain undefined, these isoforms continue to gain scientific interest. To date, X-ray crystal structure of only one hCA-RP (hCA-RP VIII) has been determined⁴³. hCA-RP VIII is highly expressed in the cerebellum⁴⁴, and has been identified as a binding partner for the inositol 1,4,5 triphosphate (IP3) receptor type 1⁴⁵.

With a sequence identity of 41%, the overall structure of hCA-RP VIII has an r.m.s.d. of 1.3 Å with the hCA II isoform. The X-ray crystal structure of hCA-RP VIII (PDB ID: 2W2J) reveals a unique glutamate rich loop (E loop; aa 24–36) that is not seen in other hCAs. The core domain of hCA-RP VIII adopts the classical architecture of other α-CAs, namely a 10-stranded central β-sheet flanked by seven short α-helices.

A sequence homology of 98% between murine (m) CA VIII and hCA-RP VIII may well form the hypothesis that over the course of evolution the loss in CA activity is coupled to a gain of a new, though unidentified, cellular function. One possibility is the modulation of biological functions via protein–protein interactions. The type 1 IP3 receptor, identified as a hCA-RP VIII binding protein, contains an electropositive IP3 binding site⁴⁶. It is possible that the electronegative surface in hCA-RP VIII, unique among hCAs, forms a charge-complementary binding site for the receptor, thereby regulating IP3-dependent Ca²⁺ release⁴⁷. Consistent with this, a protein-interaction function has been reported for the CA like domain in receptor protein tyrosine phosphatase β which serves as the contact in binding site⁴⁸.

Stability of α-CAs

Stability is an important factor that influences the practical application of a protein. Resistance to various environmental stresses including heat, proteolytic degradation and high concentrations of denaturing agents (e.g. sodium dodecyl sulfate, acid, urea and guanidine-hydrochloride) are some of the factors that define the stability of any protein. Mostly, thermal and chemical stability are strongly correlated^49,50, but this trend is not a valid assumption in many cases^51,52. Previous studies involving denaturation and folding of CA have provided an insightful understanding of its folding pathway⁸.

Cytosolic hCAs: I and II

Nearly 30 years after the discovery of CA in erythrocytes⁵³, the first detailed denaturation studies on hCAs I and II with concentrated solutions of urea and guanidine-hydrochloride (GuHCl) were conducted⁵⁴. These and later experiments revealed that isoforms from the same species have varying midpoint concentrations of inactivation (C_m) for GuHCl: 1.5 M for hCA I versus 0.9 M for hCA II⁵⁵. This also coincides with the same isoform from different species having different stability: bovine (b) CA II denatures in GuHCl at a C_m of 1.6 M⁵⁶ and has also been shown to be less susceptible to denaturation at high pH than hCA II^57,58. With the sequence homology of hCA I, hCA II and bCA II being ~60%, the differences seen in stability between these isoforms seem to arise from as little as two to three amino acid substitutions in the primary sequence. Several studies examining the affect of point mutations in the stability of hCA II in GuHCl show varying destabilization in these variants with C_m values ranging from 0.9 M to <0.1 M^59–64.

Early studies involving GuHCl denaturation also revealed that the unfolding pathway of hCA I, hCA II and bCA II occurs in two well-defined transition states via a stable molten-globule intermediate composed of the central β-strands^54,65. Studies involving mutagenesis of residues in this hydrophobic core into radio-labelled cysteines revealed that some portion of this region of hCA II remained folded up to solubility limit of GuHCl (~6 M)^59,66,67. Interestingly, the same point mutations mentioned above that showed varying stability of the native structure had little to no effect on the stability of the molten globule state and, in some cases, actually increased the C_m value for the intermediate^59–64. Truncation of the first five amino acid residues in the N-terminus of hCA II results in a moderate destabilization (ΔC_m = 0.5 M compared to that of wild-type hCA II) of the native state⁶⁴. Further truncation of the N-terminus up to 24 residues does not significantly destabilize the native or the molten globule state of the enzyme. This suggests that there are only one or more residues within the first 24 amino acids that interact(s) with the rest of the protein and contribute(s) minimum stabilization energy (~5 kcal/mol)⁶⁴. In contrast, digestion of the first three C-terminus residues of hCA I with carboxypeptidase revealed a significant decrease in stability from a C_m of 1.5 M GuHCl for the native enzyme to 0.6 M for the truncated variant⁶⁸.

The metal in the active site of CAs also affects the stability of the enzyme. According to the differential scanning calorimetry (DSC) studies performed on the native and apo-form of hCA II, the enzyme is destabilized by approximately 7°C when comparing the melting temperature (T_m) of the two forms (58°C versus 51°C for native and apo-hCA II, respectively), resulting in a destabilization of about 12 kcal/mol⁶⁹. Cobalt can replace the zinc in the active site of hCA II without major structural changes or significant loss of activity and stability^70,71 whereas, the C_m-value decreases by approximately 0.2 M GuHCl for cobalt-substituted hCA I and bCA II⁷² as compared to hCA II.

Studies involving tryptophan fluorescence confirmed the absence of a molten-globule intermediate in the denaturation pathway of hCA II with a C_m value of 4.4 M urea⁷³. The authors predict that the difference in denaturation between urea and GuHCl is due to the ionic character of GuHCl. The high ionic strength of NaCl may help to weaken the ionic interactions in the native state of hCA II which could explain the similar results seen between denaturation using GuHCl and urea with high salt concentrations.

Circular dichroism (CD) studies performed on hCA I and II suggest approximately 20% α-helix characterization below pH 4, which is much higher than that of the protein near neutral pH⁷⁴. During the second transition, measurements of the intrinsic viscosity (η) of a solution of bCA II suggests that only partial denaturation occurs at pH 2 (η = 8.4 cm³/g)⁷⁵, unlike that seen with GuHCl or urea (η = 29.6 cm³/g) which is consistent with the viscosity of a solution containing random coil characteristics (η = 29 cm³/g)⁷⁶. This partial denaturation in acidic conditions was also observed in hCA I and II with the stability showing a strong dependence on the ionic strength of the buffer^58,77. Incomplete denaturation of CA in acidic conditions could be due to electrostatic repulsions that fail to overcome hydrophobic forces, salt bridges and other favourable interactions.

Other cytosolic hCAs: III, VII and XIII

The stability of the other hCAs has not been as extensively studied as hCAs I and II, but what has been reported to date in the literature reveals some similarities and interesting differences between the isoforms. CD studies on bCA III showed a similar profile to that of hCA II, with a molten-globule intermediate occurring at a C_m value of 1.0 M GuHCl and a completely unfolded state at a C_m of 2.6 M GuHCl⁷⁸. Although bCA III contains five free cysteine residues that could form inter- and/or intra-molecular disulphide bridges during the unfolding process, the CD spectra of bCA III in reducing conditions were similar to those without dithiothreitol (DTT). This suggests that these cysteines do not form linkages during denaturation. Interestingly, hydratase and esterase activity studies on CA III isolated from mice (mCA III) at various ages revealed that the mCA III from older mice shows a significant tolerance to thermal denaturation compared to the young mice⁷⁹. This is believed to be a result of glutathiolation resulting in an unusual disulphide linkage between cysteine residues and glutathione and could provide protection against the highly oxidative environment⁷⁹.

The X-ray crystal structure of hCA VII shows a disulphide bond between residues 54 and 178 (hCA VII numbering)⁸⁰. However, the authors were careful to note that these two cysteines are not conserved in other α-CAs and that disulphide bonds are rare among cytosolic proteins. It was concluded, therefore, that this disulphide bond could be a result of the oxidizing conditions that arise during protein handling. As of present, there have been no stability studies done with hCA VII but, based on the sequence identity to hCA I (51%), hCA II (56%) and hCA III (53%) (Table 2) similar stability characteristics could be assumed.

Recently, the heat dependency of hCA XIII denaturation has been reported⁸¹. It was shown by DSC studies that hCA XIII undergoes a two-state denaturation phase change from native to a completely unfolded state, similar to those previously reported for hCA I⁸² and hCA II^69,83, with a T_m at approximately 59°C. It was also seen that when complexed with a tight-binding inhibitor of CA, ethoxyzolamide, the T_m increased to 72°C, a common trend seen with other inhibitors of hCA II⁸.

The extracellular hCAs: IV, VI, IX, XII and XIV

The X-ray crystal structure of hCA IV⁸⁴ revealed that the presence of two disulphide linkages between 6-11G and 23-203 (hCA IV numbering) may contribute to its stability in 5% SDS^85,86. The latter disulphide linkage stabilizes an important loop in the active site containing Thr199, which hydrogen bonds to and orients the zinc-bound hydroxide for catalysis⁸⁴. A disulphide bridge between residues 23 and 203 was engineered in hCA II. Additionally, Cys206 was mutated to Ser in order to avoid 23–206 disulphide formation. An enhancement in stability of this triple mutant was observed: ▵C_m = 0.8 M GuHCl⁸⁷ and ▵T_m = 13°C (unpublished).

This disulphide (23–203) linkage is conserved in hCAs VI, IX, XII and XIV^37,88–90, but these isoforms are more sensitive to SDS than hCA IV. This suggests that the additional disulphide bridge between residues 6 and 11G in hCA IV accounts for the enhanced SDS-resistance⁹⁰. Additionally, the X-ray crystal structure of hCA IX reveals a dimeric complex that is linked together via an intermolecular disulphide bridge at residue 41 (hCA IX numbering)⁸⁸. The implications on stability of this linkage are not yet fully understood, but hCA IX has been associated with tumour cells which are surrounded by a highly acidic environment compared to those of other hCAs⁹¹.

The CA-RPs: hCAs VIII, X and XI

Unfolding studies on hCA-RP VIII with GuHCl showed that this isoform, like hCA II, unfolds in two distinct transitions, but was more sensitive to GuHCl chemical denaturation than hCA II (C_m = 0.4 M for hCA-RP VIII, and C_m = 0.9 M for hCA II)⁴¹. Based on near-UV CD experiments, it was concluded that the destabilization of hCA-RP VIII could be due to an extended N-terminus, giving it a less compact tertiary structure as compared to hCA II. The sensitivity of hCA-RP VIII to GuHCl could be due to the high ionic character found on the surface (E-loop and α3-β15 loop), as the proposed mechanism of GuHCl denaturation occurs via disruption of ionic charges on the surface of hCA II⁷³. Additionally, the lack of a metal coordinating to the active site has shown to have destabilizing effects in both hCA II and bCA II^69,92. Stability studies and high-resolution structures of hCA-RPs X and XI are not yet available, but it may be predicted that the lack of a metal-coordinating ion in the active site of these proteins would have similar destabilizing effects as those seen with hCA-RP VIII, apo-hCA II and bCA II.

Discussion

Recently, the determination of crystal structures for all the catalytically active α-CAs (i.e. hCA I⁹³, hCA II²⁸, hCA III³³, hCA IV⁸⁴, mCA VA³⁴, hCA VI (unpublished, PDB ID 3FE4), hCA VII (unpublished, PDB ID 3MDZ), hCA IX⁸⁸, hCA XII⁸⁹, hCA XIII³⁸ and mCA XIV⁹⁰ will provide a better understanding of the mechanisms of inhibition of different hCAs. This has initiated significant advances in the development of CAIs and their administration in humans⁹⁴.

Most tumours experience a structurally and functionally disturbed microcirculation of oxygen which pathophysiologically causes an inadequate supply of oxygen (a condition called hypoxia)^5,95–100. There is evidence that hCA IX expression allows tumours to acclimate to a hypoxic microenvironment, promoting tumour cell proliferation, and that hCA IX expression is related to poor survival in patients^39,101. hCA IX is a transmembrane protein with a extracellular hCA catalytic domain. This domain is very similar to hCA II and acts as an off-target for most CA inhibitors (CAIs). The only known structure of hCA IX (PDB ID: 3IAI)⁸⁸ has an r.m.s.d. of 1.5 Å with hCA II (PDB ID: 3KS3)²⁸. However, the existence of certain amino acid differences on the surface, referred to as the “selective pocket”, in hCA IX¹⁰² could be exploited for design of isoform specific CAIs. Apart from the catalytic CAs, determination of crystal structure of hCA-RP VIII (PDB ID: 2W2J)⁴³ has reinforced the efforts to study the other two hCA-RPs and investigate the possible role of these acatalytic CA isoforms.

The biophysical properties of CAs that contribute to the stability and folding pathway have been extensively studied. While these studies have mainly focused on hCAs I and II, the conclusions can be applied to other protein families in which these characteristics remain elusive. Further research is needed to fully characterize the stability of several human isoforms, namely the extracellular hCAs (IV, VI, IX, XII and XIV) as well as the mitochondrial hCAs (VA and VB). All of these isoforms experience unique environments compared to those of the cytosolic hCAs (I, II, III, VII and XIII).

The favourable properties of hCA II (high kinetic parameters, easy expression, high solubility, intermediate heat resistance) have made it an attractive candidate for numerous industrial applications⁸³; however, there are few prokaryotic extremophilic CAs (such as SspCA and SazCA) recently discovered in Sulfurihydrogenibium yellowstonense that show comparable enzymatic activity and higher thermostability^103,104. There is an increasing industrial interest in using hCA II as a bio-catalyst for carbon sequestration of flue-gas from coal-fired power plants. Also, there are established protocols utilizing CA found in algae to capture carbon dioxide and convert it into biofuels and other valuable products^105,106. There is also interest in using apo-CAs as a bio-sensor for zinc and other transition metals in sea water or human serum¹⁰⁷.

For industrial applications, small improvements in stability without detriment to yield, activity or solubility, can accelerate the development of hCA II as a better bio-catalyst. Use of the free enzyme in solution can also have disadvantages, as the low-stability can limit recycling and cost-efficiency in an industrial setting¹⁰⁸. As such, there are numerous studies underway to enhance the stability of hCA II while also retaining its characteristic high catalytic efficiency. One such study deals with mutating some residues to Cys in wild-type hCA II in order to form disulphide linkages⁸⁷. Figure 6 shows regions in hCA II where Cys residues can possibly be engineered, based on their presence in other hCAs. Other studies include immobilization of hCA II on a variety of surfaces^108,109 and directed-evolution of the enzyme involving mutagenesis of surface hydrophobic residues into hydrophilic moieties⁸³. Further research is needed to maximize the stability of hCA II in a wide array of environments without the loss of catalytic efficiency for industrial use.

Figure 6.  Stereo cartoon-surface representation of hCA II (gray) showing the analogous positions of cysteines (green) present in all the catalytic hCAs. Active site Zn²⁺ is shown as a magenta sphere.

Declaration of interest

The work has been supported by Alumni Fellowship Award, Grinter Award and Medical Guild Research Incentive Award from University of Florida (MA), HHMI Science for Life (BK) and National Institutes of Health grant GM25154 (RM). The authors report no conflict of interest.