Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase
aDepartment of Physics, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea, and bDepartment of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA
*Correspondence e-mail: email@example.com
Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3−) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (kcat and kcat/Km) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure–function relationships of many other biologically important enzymes.
Keywords: carbonic anhydrase II; metalloenzymes; active-site mutation; active-site water dynamics; zinc ion; X-ray crystallography; enzyme mechanism; structural biology.
Enzymes greatly enhance the catalytic rates of biochemical reactions compared with their uncatalyzed counterparts and are therefore essential to speed up biochemical processes (Jencks, 1987; Fersht, 1999; Frey & Hegeman, 2007). Enzyme active sites provide highly optimized microenvironments for their specific substrates by providing reactive groups such as nucleophiles or acids/bases that stabilize the transition state. Consequently, changes in the active-site residues can have large effects on enzyme activity. However, direct prediction of the impact of a single mutation on the activity of an enzyme remains challenging owing to the lack of precise correlations between the structure of the protein and its function at atomic resolution (Ishida, 2010). In this study, we describe the effect of a single amino-acid variation on a prototypical enzyme, human carbonic anhydrase II (CA II), by correlating its high-resolution reaction-intermediate structures with the measured kinetic parameters. Human carbonic anhydrases are well suited to serve as a model system for our study because their structures and active sites are well defined, and their overall enzymatic mechanism is fairly straightforward and has been studied extensively (Krishnamurthy et al., 2008).
Human carbonic anhydrases catalyze the reversible hydration/dehydration of CO2/HCO3− (Davenport, 1984; Christianson & Fierke, 1996; Chegwidden et al., 2013; Frost & McKenna, 2013; Supuran & De Simone, 2015). In the CO2-hydration direction, the first step of catalysis is the conversion of CO2 into HCO3− via the nucleophilic attack of a zinc-bound hydroxide. This reaction is followed by the displacement of the zinc-bound HCO3− by a water molecule (equation 1, where E stands for the enzyme; Silverman & Lindskog, 1988). The second step involves the transfer of a proton from the zinc-bound water to bulk solvent, regenerating the zinc-bound hydroxide (equation 2, where B stands for a general base: either a water or a proton-shuttling residue).
In CA II, the active-site zinc is located at the base of a 15 Å deep cleft and is tetrahedrally coordinated by three histidines (His94, His96 and His119) and a zinc-bound water (WZn) [Fig. 1(a)] (Christianson & Fierke, 1996). The active-site cavity is further subdivided into two distinct faces consisting of hydrophilic and hydrophobic residues. The hydrophilic face (Tyr7, Asn62, His64, Asn67, Thr199 and Thr200) of the active site coordinates the hydrogen-bonded water network (W1, W2, W3a and W3b) that connects the zinc-bound water to His64, the proton-shuttling residue [Fig. 1(b)] (Steiner et al., 1975; Tu et al., 1989; Nair & Christianson, 1991; Fisher et al., 2005, 2010; Fisher, Maupin et al., 2007; Fisher, Tu et al., 2007; Maupin & Voth, 2007; Silverman & McKenna, 2007; Zheng et al., 2008). It is known that the proton-transfer process is the rate-limiting step in CA II catalysis (Silverman & McKenna, 2007).
The hydrophobic face (Val121, Val143, Leu198, Val207 and Trp209) is located adjacent to the zinc-bound hydroxide and is responsible for substrate binding (Liang & Lipscomb, 1990; Domsic & McKenna, 2010). Leu198, Trp209 and Val121 constitute the mouth and sides of the hydrophobic pocket, while Val143 comprises the base of the hydrophobic pocket. A water molecule termed the `deep water' (WDW) is located at the mouth of this pocket and forms van der Waals contacts with Leu198 and Trp209. WDW occupies the pre-catalytic association site for substrate and is displaced by one of the O atoms of CO2 during binding (Domsic et al., 2008). The hydrophobic pocket residues are highly conserved and are known to be critical for CO2 sequestration, although they do not directly interact with the CO2 molecule.
Between the hydrophilic and hydrophobic sides of the active site, a cluster of ordered waters has recently been identified located near the active-site entrance, termed the entrance-conduit (EC) waters [Fig. 1(c)] (Kim et al., 2018). This ordered water ensemble connects the active site to the external bulk solvent, creating a pathway where water, substrate and product can interchange and interact with bulk solvent. These EC waters are believed to be involved in water replenishment during catalysis, displacing the zinc-bound bicarbonate and restoring the proton-transfer water network.
In order to elucidate the catalytic details of the active site of CA II, several mutational studies have been performed. Most of these variants have been focused at the zinc ion-binding site (Alexander et al., 1993; Kiefer et al., 1993; Ippolito & Christianson, 1994; Lesburg & Christianson, 1995; Huang et al., 1996; Lesburg et al., 1997), the hydrophilic side (proton-transfer pathway; Behravan et al., 1990; Krebs, Ippolito et al., 1993; Xue et al., 1993; Ippolito et al., 1995; Huang et al., 2002; Tu et al., 2002; Fisher et al., 2005; Zheng et al., 2008; Turkoglu et al., 2012; Mikulski et al., 2013; Aggarwal et al., 2014) and the hydrophobic pocket (CO2-binding site; Alexander et al., 1991; Fierke et al., 1991; Nair et al., 1991; Krebs, Rana et al., 1993; West et al., 2012; Nair & Christianson, 1993). In the hydrophobic pocket, a series of mutational studies have been performed targeting the Val121, Val143 and Leu198 residues. It was found that CA catalysis is severely compromised (an ∼104–105-fold decrease) when the deep water WDW is displaced by replacement of the relevant amino acid by one with a larger side chain (for example, replacement of Val143 by Phe or Tyr), leading to a substantial blockage of CO2 binding. On the other hand, some of the point mutations, such as Val121 to Ala, Val143 to Ile and Leu198 to Glu, do not directly displace WDW and the CA catalysis is only moderately compromised (a fewfold to a 20-fold decrease). Understanding these moderate effects is most challenging as the overall structures and active sites show little deviation when compared with native CA II. It is expected that delicate perturbations are introduced at the level of intermediate structures during the moderately modified CA catalysis.
In this study, we investigate one of the most challenging cases and describe the subtle effects brought on by a Val143 to Ile (V143I) mutation. As shown in Table 1, the V143I variant shows an approximately tenfold decrease in kcat/Km, while kcat remains almost the same as that for native CA II. Structural analysis was performed by comparing the catalytic intermediate states of native and V143I CA II, which were obtained by cryocooling protein crystals under four different CO2 pressures [ranging from 0 (no CO2 pressurization) to 15 atm]. The intermediate states are henceforth referred to as native-0atm (PDB entry 6km3), native-7atm (PDB entry 6km4), native-13atm (PDB entry 6km5) and native-15atm (PDB entry 6km6) and as V143I-0atm (PDB entry 6klz), V143I-7atm (PDB entry 6km0), V143I-13atm (PDB entry 6km1) and V143I-15atm (PDB entry 6km2), respectively. Based upon the structural modifications, we successfully estimated the alterations in the reaction rate constants and the corresponding free-energy profiles in the CA II enzymatic mechanism. This study systematically reveals how a single point mutation influences an enzyme's catalytic pathway at the atomic level, leading to an estimation of the kinetics governing its individual mechanistic steps.
‡From Fierke et al. (1991).
§From West et al. (2012).
Our X-ray studies (methods are reported in the supporting information) revealed that the overall protein backbones (tertiary structures) of the native and V143I CA II structures were very similar, with Cα–Cα r.m.s.d. values of less than 0.14 Å (Supplementary Tables S1 and S2). However, careful structural analysis successfully established subtle but clear changes in the active site (CO2-binding site, proton-transfer pathway and water-replenishment pathway; EC waters). The key bound water molecules in native and V143I CA II are listed in Supplementary Tables S3 and S4.
2.1. CO2-binding site around the zinc ion
Fig. 2 shows the CO2-binding site and crucial water molecules (WZn/WDW/WI/WI′/W1) in the vicinity of the zinc ion. In native CA II [Figs. 2(a)–2(d)], the water molecules (WZn/WDW/W1) around the zinc ion are initially well ordered at 0 atm CO2 pressure. At higher CO2 pressures of 7–15 atm, electron density for the CO2 molecule becomes apparent, displacing the deep water (WDW). Concurrently, two intermediate waters WI and WI′ emerge near Thr200, while W1 disappears at higher CO2 pressures. The distance between WI and W2 is ∼4.7 Å, suggesting that the hydrogen-bonded water network that facilitates proton transfer is disrupted when the CO2-binding site is fully occupied.
On the other hand, the active site of V143I CA II shows noteworthy modifications. The most striking difference is that HCO3− is stabilized and is observable at 0 atm CO2 pressure with an estimated occupancy of ∼20% [Fig. 2(e)]. In the absence of experimentally introduced CO2, it is likely that the captured HCO3− is converted from CO2 absorbed into the crystal from ambient air. As the CO2 pressure increases, the HCO3− occupancy increases to 54% at 13 atm and subsequently decreases slightly to 48% at 15 atm, with increased CO2 occupancy [Figs. 2(f)–2(h)]. It is likely that the observed decrease in the HCO3− occupancy at 15 atm is owing to steric hindrance from the bound CO2 molecule. When superimposed with the previously reported coordinates of HCO3− bound to native CA II (PDB entry 2vvb; Sjöblom et al., 2009), the HCO3− position observed in V143I CA II shows noticeable deviations. The HCO3− molecule is tilted by 35° with respect to the plane containing WZn, CO2 and HCO3− in native CA II (Supplementary Fig. S1). In addition, the central C atoms of the two superimposed HCO3− molecules in native and V143I CA II are separated by 0.5 Å.
Unlike the HCO3− molecule, the CO2 molecule is less stable in V143I CA II. For example, CO2 shows almost full occupancy at 7 atm in native CA II, while no CO2 is visible at 7 atm in V143I CA II, which instead shows the appearance of WDW [Figs. 2(b) and 2(f)]. The CO2 molecule appears at higher pressures (13 and 15 atm) with a decreased occupancy of ∼50% [Figs. 2(g) and 2(h)]. The bound CO2 is tilted by ∼6° and is situated closer to the zinc ion by 0.34 Å compared with that in native CA II (Supplementary Fig. S1). The distance between the end carbon (Cδ1) of Ile143 and the CO2 molecule is only 3.0–3.2 Å, and this steric disruption seems to affect the critical interactions between the CO2 molecule and the hydrophobic pocket, thereby destabilizing it in the active site. It is worth noting that in contrast to native CA II, the bound CO2 molecule in V143I CA II is distorted from the plane defined by the bound HCO3− molecule, and this seems to be detrimental to the efficient conversion of CO2 to HCO3−.
Finally, the two intermediate waters WI and WI′ show different behaviour in V143I CA II. The intermediate water WI is visible even in 0 atm V143I CA II but is not present in 0 atm native CA II [Figs. 2(a) and 2(e)]. However, it is observed that the electron densities of the two intermediate waters were less defined than in native CA II [Figs. 2(f)–2(h)]. These `weaker' intermediate waters were accompanied by the presence of W1 at all pressures (0–15 atm). It is likely that these weak intermediate waters are related to the perturbed structures and dynamical motions of the EC waters, as explained later.
2.2. Proton-transfer pathway
Fig. 3 shows the proton-transfer pathway including the water network (W1/W2/W3a/W3b) and His64. In native CA II, the water network is initially well ordered at 0 atm CO2 pressure [Fig. 3(a)]. As the CO2 pressure increases, W1 disappears and intermediate waters (WI and WI′) emerge instead [Figs. 3(b)–3(d)]. The W2 water, which transfers a proton from W1 to His64, shows an alternative position denoted W2′. Considering the steric hindrance between His64in and W2′, it seems that the presence of W2′ pushes His64 towards the `out' conformation (away from the water network). Indeed, His64 shows a net movement from the `in' to the `out' conformation as W2′ becomes prominent at higher CO2 pressures. W3a shows little change, but W3b shows an alternative water position, W3b′, and is found to interact with one of the entrance waters WEC1 (and its alternative position W′EC1).
Similarly, in V143I CA II W2 shows the same alternative position W2′, and His64 shows the same `in' to `out' flip, with similar occupancies as observed in native CA II with increasing CO2 pressure [Figs. 3(e)–3(h)]. W3b and WEC1 also show the same alternative positions, although their electron densities are slightly weaker. As the distance between W2 and the N atom (Nδ1) of His64in is relatively long (3.3 Å), efficient proton transfer seems to depend on the dynamical motions of W2/W2′ and His64in/His64out. These motions are quite similar in native and V143I CA II and therefore proton transfer is not significantly impacted in the variant.
2.3. Water-replenishment pathway
Fig. 4 shows the water-replenishment pathway consisting of the ordered EC waters (WEC1/WEC2/WEC3/WEC4/WEC5). In native CA II, the five WEC waters are well ordered at 0 atm CO2 pressure [Fig. 4(a)]. As the CO2 pressure increases, WEC1 shows an alternative position W′EC1, and WEC2 shifts to an alternative position W′EC2 [Figs. 4(b)–4(d)]. These dynamical motions of WEC1 and WEC2 are accompanied by the emergence of the intermediate waters WI and WI′. The two intermediate waters are located deep within the entrance conduit near the active site and are transiently stabilized via hydrogen bonding to several WEC waters (WEC2, WEC3 and WEC5 and their alternative positions). The short distance (2.2 Å) between WI and WI′ suggests that WI′ can rapidly shift to the WI position as WI refills the vacant water positions (W1/WZn/WDW) during catalysis. Previous studies suggest that the intermediate waters (WI and WI′) play a critical role in the rapid replenishment of the active-site water network during catalysis and therefore could influence the overall catalytic rate (Kim et al., 2018).
Among the five WEC waters in V143I CA II, WEC2 is located close to the mutated residue Ile143. Indeed, the WEC1 to WEC4 waters show similar structures and dynamical motions as in native CA II, but WEC2 shows a significantly distinct behaviour. Initially, at 0 atm CO2 pressure, the WEC2 water shows multiple alternative positions (W′′EC2, W′′′EC2 and W′′′′EC2) [Fig. 4(e)]. As the CO2 pressure increases, these alternative positions disappear and both the WEC2 and W′EC2 waters are observed instead [Figs. 4(f)–4(h)]. Compared with native CA II, W′′EC2, W′′′EC2 and W′′′′EC2 are new alternative positions that are observed only at 0 atm CO2 pressure in V143I CA II. Along with the perturbed dynamical motions of the WEC2 water, much weakened electron densities of the intermediate waters WI and WI′ are observed. It should be noted that among the five EC waters, WEC2 is unique in that it interacts with all three of the key waters, W1, WI and WI′. Therefore, the fact that the structures and dynamical motions of the WEC2 waters are significantly perturbed in V143I CA II is likely to account for the stabilization of W1 but the destabilization of the two intermediate waters.
Another interesting aspect of WEC2 is that its alternative position W′′′′EC2 is situated close to the bound HCO3−. The distances between W′′′′EC2 and the closest O atom and the C atom of HCO3− are 1.4 and 2.5 Å, respectively (Supplementary Table S5). Since W′′′′EC2 is too close to the bound HCO3−, W′′′′EC2 and HCO3− cannot coexist in tandem. Indeed, W′′′′EC2 is only visible in V143I CA II at 0 atm, when the HCO3− occupancy is low, and disappears as the HCO3− occupancy increases at higher CO2 pressures [Figs. 4(e)–4(h)]. It seems that the relationship between W′′′′EC2 and HCO3− is analogous to the relationship between WDW and CO2. In native CA II, the hydrophobic cavity produces an electrostatic environment in which the deep water (WDW) can be locally stabilized around the zinc ion and then replaced with one of the O atoms of CO2 upon CO2 binding. In V143I CA II, the altered hydrophobic cavity owing to the V143I mutation produces a slightly different electrostatic environment in which an additional water position (W′′′′EC2) can be locally stabilized around the zinc ion and subsequently replaced with one of the O atoms in HCO3− during CA II catalysis. The release of the W′′′′EC2 molecule upon HCO3− binding seems to reduce the entropic cost of the process, analogous to the release of WDW upon CO2 binding in native CA II. Thus, it is likely that the altered multiple conformations of WEC2 allow HCO3− to bind more easily and firmly in a tilted configuration at the active site.
In this study, we have successfully identified the `fine' structural changes in the CA II intermediates induced by a single-residue mutation at the active site. The V143I mutation in CA II produces steric hindrance and induces subtle changes in the electrostatic environment of the active site. The resulting effects on the CA II intermediates can be summarized as follows: (i) the dynamical motions and the allowed configurations of CO2 are slightly restricted and the binding affinity of HCO3− is increased with a distorted configuration and (ii) the EC water network in the water-replenishment pathway is restructured, while (iii) the proton-transfer dynamics are mostly unaffected. These detailed structural insights can now be used to assess the modifications in the reaction rate constants (k1, k−1, k2 and k3) and the corresponding free-energy profiles during the CO2-hydration reaction of CA II.
Firstly, the V143I mutation restricts the configurational freedom of the CO2 molecule within the hydrophobic pocket of CA II. Previous mutational studies on hydrophobic pocket residues (Val121 and Val143) suggest that the hydrophobic pocket of native CA II is involved in `ushering' the CO2 molecule to the zinc hydroxide in the active site, but does not directly interact with the CO2 molecule to hold it in a specific orientation. Therefore, it is most likely that the CO2 molecule in aqueous solution is guided to the hydrophobic pocket of CA II but retains some degrees of freedom with regard to the acceptable configurations (positions and orientations) around the zinc hydroxide that eventually facilitate its rapid conversion into HCO3−. In the V143I variant, although the hydrophobicity of the active-site pocket is increased, the added methyl group appears to sterically restrict the number of favourable configurations and the dynamical motions accessible to the CO2 molecule within the cavity. This estimation is supported by the crystallographic observation that the CO2 molecule is distorted towards the zinc ion, tilted by ∼6° and is destabilized with lower occupancies in the V143I variant. Consequently, interconversion from CO2 to HCO3− becomes less efficient, leading to a reduced k1 (k1V143I < k1native). The lower k1 value also implies an enhanced activation-energy barrier for the step [Arrhenius relationship: k1(T) = Aexp(−E1/RT), where A is a pre-exponential constant, R is the molar gas constant, T is the absolute temperature and E1 is the activation energy] (Fig. 5).
Secondly, the V143I mutation induces a slightly different electrostatic environment in the hydrophobic cavity, thereby altering the location and dynamics of the WEC2 water. It seems that one of these altered WEC2 waters (W′′′′EC2) increases the binding affinity of the HCO3− molecule in the active site. This stronger binding of HCO3− suggests that the free energy of the enzyme–product (EZnHCO3−) complex is lowered in the V143I variant (Fig. 5). In conjunction with the larger activation energy for the k1 reaction, it can be deduced that the activation energy for the reverse reaction k−1 is increased even further, leading to a reduced k−1 value (k−1V143I < k−1native). On the other hand, the alterations of the WEC2 water in the V143I variant make the intermediate waters (WI and WI′) slightly less stable, therefore possibly slowing down the replenishment of the active-site water network and HCO3− dissociation. This results in a reduced k2 value (k2V143I < k2native).
Thirdly, the V143I mutation seems to have little effect on the kinetics of the proton-transfer reaction. It is observed that W1 is more stabilized in the V143I variant intermediates. It should be noted that the intermolecular proton transfer can occur via the fully established water network WZn→W1→W2→His64. This implies that if a mutation induces the destabilization of W1, proton transfer could be significantly perturbed. However, stabilization of W1 as in the V143I variant does not necessarily suggest a faster proton-transfer process. Rather, it is the dynamical motions of W2 and His64 that have a more critical influence on the proton-transfer rate. Our results suggest that both W2 and His64 show very similar dynamical motions. Taken together, it seems that the overall proton-transfer rate k3 is not significantly altered in the V143I mutant (k3V143I ≃ k3native).
The interplay between the modified reaction rate constants, as discussed above, now allows us to determine the effect of the V143I mutation on the measured kinetic parameters (kcat and kcat/Km; Nair et al., 1991; Krebs, Ippolito et al., 1993). The steady-state kinetic parameters for the CO2-hydration reaction are listed in Table 1. The kcat value shows little change, but the second-order rate constant kcat/Km shows an approximately tenfold decrease in the V143I variant. The parameter kcat contains rate constants from the initial enzyme–substrate complex through the remaining steps, including proton transfer. Therefore, for the proposed mechanistic scheme (equations 1 and 2), kcat can effectively be represented as kcat = k2k3/(k2 + k3). On the other hand, the ratio kcat/Km contains rate constants for the initial association of the substrate CO2 through the dissociation of the product HCO3−. Hence, kcat/Km only contains rate constants from equation 1 (and not equation 2) and is represented as kcat/Km = k1k2/(k−1 + k2).
It is known that in native CA II the HCO3−-dissociation process (k2) is much faster than the reverse interconversion from HCO3− to CO2 (k−1) and the proton-transfer rate (k3), i.e. k2native ≫ k−1native, k2native ≫ k3native, with k2native ≃ 10k−1native, k2native ≃ 15k3native and k−1native ≃ 1.5k3native (Table 1). Combining this information with the insights gleaned from our study (k1V143I < k1native, k−1V143I < k−1native, k2V143I < k2native and k3V143I ≃ k3native), we can estimate the effect of the V143I point mutation on the observed kinetic parameter kcat in the following way. In the native state [kcat]native = k2nativek3native/(k2native + k3native) ≃ k3native (using k2native ≫ k3native), indicating that the turnover rate in the native is almost the same as the proton-transfer rate and that the proton-transfer process is the rate-limiting step. In the V143I variant [kcat]V143I = k2V143Ik3V143I/(k2V143I + k3V143I) ≃ k3native[k2V143I/(k2V143I + k3V143I)] (using k3V143I ≃ k3native), and the experimental measurement (Table 1) suggests that [kcat]V143I ≃ [kcat]native ≃ k3native, implying that [k2V143I/(k2V143I + k3V143I)] ≃ 1, or k2V143I ≫ k3V143I. This result indicates that HCO3− dissociation (k2) remains faster than proton transfer (k3) in the V143I variant and that the proton-transfer process is still the rate-limiting step in the V143I variant. The negligible reduction in k2V143I suggests that its activation energy is not significantly increased (Fig. 5).
The second-order rate constant kcat/Km shows the following relationship in the native state: [kcat/Km]native = k1nativek2native/(k−1native + k2native) ≃ k1native (using k2native ≫ k−1native). This indicates that [kcat/Km]native mostly reflects the CO2-binding step and its interconversion to HCO3−. On the other hand, in the V143I variant both the dissociation of HCO3− (k2) and the reverse interconversion (k−1) from HCO3− to CO2 are retarded (k2V143I < k2native and k−1V143I < k−1native), leaving the relation k2V143I ≫ k−1V143I unresolved. However, the relation k2V143I ≫ k3V143I ≃ k3native obtained from the kcat analysis above suggests that k2V143I is still an order of magnitude larger than k3native, while k−1V143I < k−1native ≃ 1.5k3native suggests that k−1V143I is comparable to or less than k3native, thereby ensuring that the relation k2V143I ≫ k−1V143I remains valid. Thus, we can estimate the kcat/Km in the V143I variant in the following manner: [kcat/Km]V143I = k1V143Ik2V143I/(k−1V143I + k2V143I) ≃ k1V143I (using k2V143I ≫ k−1V143I). This estimation indicates that the reduced [kcat/Km]V143I is mostly owing to the decrease in k1, reflecting the slower CO2 binding and interconversion to HCO3− in the V143I variant. It should be noted that the reduction in the k−1 value has little effect on [kcat/Km]V143I in the direction of CO2 hydration, as long as the reduction in k2 is small enough to keep the relation k2V143I ≫ k−1V143I valid. However, it is expected that the reduction in the k−1 value would have significant consequences for the HCO3−-dehydration direction.
Finally, considering that kcat ≃ k3 and [kcat/Km] ≃ k1 in both native and V143I CA II, the Michaelis constant Km is expressed in the following way: Kmnative = k3native/k1native and KmV143I = k3V143I/k1V143I. Consequently, KmV143I > Kmnative can be obtained using the estimated relationships k1V143I < k1native and k3V143I ≃ k3native. The relationship shows that the substrate concentration needed to reach half of the maximum reaction velocity is larger in V143I CA II mainly owing to the slower CO2 binding and interconversion to HCO3−.
Although our study was performed for a single point mutation within the hydrophobic pocket (V143I), our approach and interpretations can be extended to arbitrary mutations in CA II. Considering the forward CO2-hydration direction, the mutation can first perturb substrate funnelling into the hydrophobic pocket via steric hindrance, thereby limiting the configurations that allow its efficient conversion into product. This influence is directly reflected in kcat/Km, but not in kcat. Secondly, the mutation can structurally distort the proton-transfer pathway by perturbing the water network or its associated stabilizing residues, and this effect is directly reflected in kcat but not in kcat/Km. Thirdly, the mutation can alter the product-dissociation process via direct steric hindrance or perturbations in the water-replenishment pathway. This influence can be intricate and is reflected both in kcat and kcat/Km. On the other hand, the mutation can affect the reverse reaction, the interconversion from product to substrate and substrate dissociation, but this has little influence on either kcat or kcat/Km in the hydration direction, as long as the reverse interconversion process is much slower than the product-dissociation process.
We systematically studied the effect of a single-residue mutation on the CA II catalytic pathway at atomic resolution. We have successfully captured the high-resolution intermediate states of the V143I variant and shown clearly that the single point mutation induces noticeable changes in substrate and product binding at the active site and in the water-replenishment pathway, but has little effect on the proton-transfer pathway. The structural information was then utilized to estimate the reaction rate constants and the free-energy profiles during the catalytic cycle, unravelling the effect of the point mutation on the altered kinetic parameters. We believe that the detailed and systematic approach in our CA II study can be extended to identify the specific roles of target amino-acid residues in many other biologically important enzymes. We also anticipate that our detailed descriptions could serve as a reference point for future theoretical and computational studies that may lead to an advanced understanding of enzyme mechanisms at the quantum-chemistry level.
5. Related literature
The following references are cited in the supporting information for this article: Emsley et al. (2010), Forsman et al. (1988), Henderson (1990), Khalifah et al. (1977), Kim et al. (2005, 2006, 2013, 2016), McPherson (1982), Murshudov et al. (2011), Otwinowski & Minor (1997) and Winn et al. (2011).
Supplementary Methods, Tables and Figures. DOI: https://doi.org//10.1107/S2052252520011008/jt5048sup1.pdf
The authors would like to thank the staff at Pohang Light Source II and Cornell High Energy Synchrotron Source for their support during data collection. CHESS is supported by the NSF and NIH/NIGMS via NSF award DMR-1829070 and the MacCHESS resource is supported by NIH/NIGMS award GM-124166. Authors contributions were as follows. CUK conceived the research. JKK, CL, SWL, JTA and AA ran the experiments. JKK and CUK analyzed the data. JKK, JTA, RM and CUK wrote the manuscript. All authors contributed to the overall scientific interpretation and edited the manuscript.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (2019R1A2C1004274).
Aggarwal, M., Kondeti, B., Tu, C., Maupin, C. M., Silverman, D. N. & McKenna, R. (2014). IUCrJ, 1, 129–135. Web of Science CrossRef CAS PubMed IUCr Journals Google Scholar
Alexander, R. S., Kiefer, L. L., Fierke, C. A. & Christianson, D. W. (1993). Biochemistry, 32, 1510–1518. CrossRef CAS PubMed Web of Science Google Scholar
Alexander, R. S., Nair, S. K. & Christianson, D. W. (1991). Biochemistry, 30, 11064–11072. CrossRef PubMed CAS Web of Science Google Scholar
Behravan, G., Jonsson, B.-H. & Lindskog, S. (1990). Eur. J. Biochem. 190, 351–357. CrossRef CAS PubMed Web of Science Google Scholar
Chegwidden, W. R., Carter, N. D. & Edwards, Y. H. (2013). The Carbonic Anhydrases: New Horizons. Basel: Birkhäuser. Google Scholar
Christianson, D. W. & Fierke, C. A. (1996). Acc. Chem. Res. 29, 331–339. CrossRef CAS Web of Science Google Scholar
Davenport, H. W. (1984). Ann. NY Acad. Sci. 429, 4–9. CrossRef CAS PubMed Web of Science Google Scholar
Domsic, J. F., Avvaru, B. S., Kim, C. U., Gruner, S. M., Agbandje-McKenna, M., Silverman, D. N. & McKenna, R. (2008). J. Biol. Chem. 283, 30766–30771. Web of Science CrossRef PubMed CAS Google Scholar
Domsic, J. F. & McKenna, R. (2010). Biochim. Biophys. Acta, 1804, 326–331. Web of Science CrossRef PubMed CAS Google Scholar
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. Web of Science CrossRef CAS IUCr Journals Google Scholar
Fersht, A. (1999). Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. New York: Freeman. Google Scholar
Fierke, C. A., Calderone, T. L. & Krebs, J. F. (1991). Biochemistry, 30, 11054–11063. CrossRef PubMed CAS Web of Science Google Scholar
Fisher, S. Z., Kovalevsky, A. Y., Domsic, J. F., Mustyakimov, M., McKenna, R., Silverman, D. N. & Langan, P. A. (2010). Biochemistry, 49, 415–421. Web of Science CrossRef CAS PubMed Google Scholar
Fisher, S. Z., Maupin, C. M., Budayova-Spano, M., Govindasamy, L., Tu, C., Agbandje-McKenna, M., Silverman, D. N., Voth, G. A. & McKenna, R. (2007). Biochemistry, 46, 2930–2937. Web of Science CrossRef PubMed CAS Google Scholar
Fisher, S. Z., Tu, C., Bhatt, D., Govindasamy, L., Agbandje-McKenna, M., McKenna, R. & Silverman, D. N. (2007). Biochemistry, 46, 3803–3813. Web of Science CrossRef PubMed CAS Google Scholar
Fisher, Z., Hernandez Prada, J. A., Tu, C., Duda, D., Yoshioka, C., An, H., Govindasamy, L., Silverman, D. N. & McKenna, R. (2005). Biochemistry, 44, 1097–1105. Web of Science CrossRef PubMed CAS Google Scholar
Forsman, C., Behravan, G., Osterman, A. & Jonsson, B.-H. (1988). Acta Chem. Scand. B, 42, 314–318. CrossRef CAS PubMed Web of Science Google Scholar
Frey, P. A. & Hegeman, A. D. (2007). Enzymatic Reaction Mechanisms. Oxford University Press. Google Scholar
Frost, S. C. & McKenna, R. (2013). Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications. Dordrecht: Springer Science+Business Media. Google Scholar
Henderson, R. (1990). Proc. R. Soc. Lond. B, 241, 6–8. CrossRef CAS Web of Science Google Scholar
Huang, C.-C., Lesburg, C. A., Kiefer, L. L., Fierke, C. A. & Christianson, D. W. (1996). Biochemistry, 35, 3439–3446. CrossRef CAS PubMed Web of Science Google Scholar
Huang, S., Sjöblom, B., Sauer-Eriksson, A. E. & Jonsson, B.-H. (2002). Biochemistry, 41, 7628–7635. Web of Science CrossRef PubMed CAS Google Scholar
Ippolito, J. A., Baird, T. T. Jr, McGee, S. A., Christianson, D. W. & Fierke, C. A. (1995). Proc. Natl Acad. Sci. USA, 92, 5017–5021. CrossRef CAS PubMed Web of Science Google Scholar
Ippolito, J. A. & Christianson, D. W. (1994). Biochemistry, 33, 15241–15249. CrossRef CAS PubMed Web of Science Google Scholar
Ishida, T. (2010). J. Am. Chem. Soc. 132, 7104–7118. Web of Science CrossRef CAS PubMed Google Scholar
Jencks, W. P. (1987). Catalysis in Chemistry and Enzymology. Mineola: Dover. Google Scholar
Khalifah, R. G., Strader, D. J., Bryant, S. H. & Gibson, S. M. (1977). Biochemistry, 16, 2241–2247. CrossRef PubMed CAS Web of Science Google Scholar
Kiefer, L. L., Ippolito, J. A., Fierke, C. A. & Christianson, D. W. (1993). J. Am. Chem. Soc. 115, 12581–12582. CrossRef CAS Web of Science Google Scholar
Kim, C. U., Hao, Q. & Gruner, S. M. (2006). Acta Cryst. D62, 687–694. Web of Science CrossRef CAS IUCr Journals Google Scholar
Kim, C. U., Kapfer, R. & Gruner, S. M. (2005). Acta Cryst. D61, 881–890. Web of Science CrossRef CAS IUCr Journals Google Scholar
Kim, C. U., Song, H., Avvaru, B. S., Gruner, S. M., Park, S. & McKenna, R. (2016). Proc. Natl Acad. Sci. USA, 113, 5257–5262. Web of Science CrossRef CAS PubMed Google Scholar
Kim, C. U., Wierman, J. L., Gillilan, R., Lima, E. & Gruner, S. M. (2013). J. Appl. Cryst. 46, 234–241. Web of Science CrossRef CAS IUCr Journals Google Scholar
Kim, J. K., Lomelino, C. L., Avvaru, B. S., Mahon, B. P., McKenna, R., Park, S. & Kim, C. U. (2018). IUCrJ, 5, 93–102. Web of Science CrossRef CAS PubMed IUCr Journals Google Scholar
Krebs, J. F., Ippolito, J. A., Christianson, D. W. & Fierke, C. A. (1993). J. Biol. Chem. 268, 27458–27466. CAS PubMed Web of Science Google Scholar
Krebs, J. F., Rana, F., Dluhy, R. A. & Fierke, C. A. (1993). Biochemistry, 32, 4496–4505. CrossRef CAS PubMed Web of Science Google Scholar
Krishnamurthy, V. M., Kaufman, G. K., Urbach, A. R., Gitlin, I., Gudiksen, K. L., Weibel, D. B. & Whitesides, G. M. (2008). Chem. Rev. 108, 946–1051. Web of Science CrossRef PubMed CAS Google Scholar
Lesburg, C. A. & Christianson, D. W. (1995). J. Am. Chem. Soc. 117, 6838–6844. CrossRef CAS Web of Science Google Scholar
Lesburg, C. A., Huang, C.-C., Christianson, D. W. & Fierke, C. A. (1997). Biochemistry, 36, 15780–15791. Web of Science CrossRef CAS PubMed Google Scholar
Liang, J. Y. & Lipscomb, W. N. (1990). Proc. Natl Acad. Sci. USA, 87, 3675–3679. CrossRef CAS PubMed Web of Science Google Scholar
Maupin, C. M. & Voth, G. A. (2007). Biochemistry, 46, 2938–2947. Web of Science CrossRef PubMed CAS Google Scholar
McPherson, A. (1982). Preparation and Analysis of Protein Crystals. Chichester: John Wiley & Sons. Google Scholar
Mikulski, R., West, D., Sippel, K. H., Avvaru, B. S., Aggarwal, M., Tu, C., McKenna, R. & Silverman, D. N. (2013). Biochemistry, 52, 125–131. Web of Science CrossRef CAS PubMed Google Scholar
Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367. Web of Science CrossRef CAS IUCr Journals Google Scholar
Nair, S. K., Calderone, T. L., Christianson, D. W. & Fierke, C. A. (1991). J. Biol. Chem. 266, 17320–17325. PubMed CAS Web of Science Google Scholar
Nair, S. K. & Christianson, D. W. (1991). J. Am. Chem. Soc. 113, 9455–9458. CrossRef CAS Web of Science Google Scholar
Nair, S. K. & Christianson, D. W. (1993). Biochemistry, 32, 4506–4514. CrossRef CAS PubMed Web of Science Google Scholar
Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. CrossRef CAS PubMed Web of Science Google Scholar
Silverman, D. N. & Lindskog, S. (1988). Acc. Chem. Res. 21, 30–36. CrossRef CAS Web of Science Google Scholar
Silverman, D. N. & McKenna, R. (2007). Acc. Chem. Res. 40, 669–675. Web of Science CrossRef PubMed CAS Google Scholar
Sjöblom, B., Polentarutti, M. & Djinovic-Carugo, K. (2009). Proc. Natl Acad. Sci. USA, 106, 10609–10613. Web of Science PubMed Google Scholar
Steiner, H., Jonsson, B.-H. & Lindskog, S. (1975). Eur. J. Biochem. 59, 253–259. CrossRef PubMed CAS Web of Science Google Scholar
Supuran, C. T. & De Simone, G. (2015). Carbonic Anhydrases as Biocatalysts: From Theory to Medical and Industrial Applications. Amsterdam: Elsevier. Google Scholar
Tu, C., Qian, M., An, H., Wadhwa, N. R., Duda, D., Yoshioka, C., Pathak, Y., McKenna, R., Laipis, P. J. & Silverman, D. N. (2002). J. Biol. Chem. 277, 38870–38876. Web of Science CrossRef PubMed CAS Google Scholar
Tu, C. K., Silverman, D. N., Forsman, C., Jonsson, B.-H. & Lindskog, S. (1989). Biochemistry, 28, 7913–7918. CrossRef CAS PubMed Web of Science Google Scholar
Turkoglu, S., Maresca, A., Alper, M., Kockar, F., Işık, S., Sinan, S., Ozensoy, O., Arslan, O. & Supuran, C. T. (2012). Bioorg. Med. Chem. 20, 2208–2213. Web of Science CrossRef CAS PubMed Google Scholar
West, D., Kim, C. U., Tu, C., Robbins, A. H., Gruner, S. M., Silverman, D. N. & McKenna, R. (2012). Biochemistry, 51, 9156–9163. Web of Science CrossRef CAS PubMed Google Scholar
Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A. & Wilson, K. S. (2011). Acta Cryst. D67, 235–242. Web of Science CrossRef CAS IUCr Journals Google Scholar
Xue, Y., Vidgren, J., Svensson, L. A., Liljas, A., Jonsson, B.-H. & Lindskog, S. (1993). Proteins, 15, 80–87. CrossRef CAS PubMed Web of Science Google Scholar
Zheng, J., Avvaru, B. S., Tu, C., McKenna, R. & Silverman, D. N. (2008). Biochemistry, 47, 12028–12036. Web of Science CrossRef PubMed CAS Google Scholar
This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.