Human carbonic anhydrase II (hCAII) is an enzyme of significant physiological importance, belonging to the carbonic anhydrase family. These enzymes catalyze the rapid conversion of carbon dioxide and water to bicarbonate (HCO₃⁻) and protons (H⁺), a reversible reaction that is crucial for various biological processes (Krishnamurthy et al. 2008). hCAII is one of the most studied and fastest enzymes in the carbonic anhydrase family, showcasing high efficiency in catalyzing this reaction (Lindskog 1997). hCAII is a zinc metalloenzyme, with a zinc ion in its active site that is essential for its catalytic activity (Kiefer and Fierke 1994). The enzyme consists of a single polypeptide chain and has a molecular weight of about 29 kDa. The zinc ion is coordinated by three histidine residues (Kiefer and Fierke 1994) and a hydroxide ion (Lipton et al. 2004) that acts as a nucleophile in the catalysis process (Lipton et al. 2004).
The primary function of hCAII, and carbonic anhydrases in general, is to maintain acid-base balance in blood and other tissues (Occhipinti and Boron 2019). The bicarbonate and protons rapidly produced from CO₂ by hCAII can then be easily transported in the bloodstream. This process is crucial in the kidneys for the reabsorption of bicarbonate and in the lungs for the release of CO₂ during respiration. hCAII also plays a role in the formation of aqueous humor in the eye (Wistrand et. al. 1986) and protecting oesophageal, gastric, and intestinal mucosa from acidity (Parkkila et. al. 1994; Lönnerholm et. al. 1985).
Mutations in the gene encoding hCAII can lead to diseases such as osteopetrosis (Sly et. al. 1983; Shah et. al. 2004), renal tubular acidosis (Nagai et. al. 1997), and cerebral calcification (Sly et. al. 1983, 1985). Inhibitors of carbonic anhydrase, such as acetazolamide (Sippel et. al. 2009; Vidgren et. al. 1990), are used clinically to treat conditions like glaucoma, epilepsy, altitude sickness, and certain types of edema, by reducing the production of bicarbonate. Due to its role in pH regulation and CO₂ transport, hCAII and its inhibitors have been studied for potential applications in managing conditions related to carbon dioxide and bicarbonate transport, as well as for carbon capture and sequestration efforts to mitigate climate change impacts (Effendi and Ng 2019). Recent research has shown that change in hCAII levels could be a factor in Alzheimer's Disease (Jang et. al. 2010). Also, mitochondria associated hCAII has been identified as a potential target for modulating neurodegeneration and age-related impairments (Pollard 2016).
Our project delves into the intricate role of enzyme dynamics in catalysis, a topic that has sparked considerable debate over the years. There has been a wealth of research focusing on how enzymes' structure and dynamics are affected by their interaction with water, a key factor in biomolecular processes. Although X-ray crystallography has proven effective in identifying structural waters, capturing the dynamic interplay within the water-enzyme network has been challenging. In our previous research, we utilized solid-state NMR relaxation dispersion analysis to investigate the dynamics at the active site of hCAII (Vasa et al. 2018, 2019; Singh et al. 2020), which is crucial for the catalytic action of carbonic anhydrases. This approach, together with chemical shift information, relaxation analysis, and molecular dynamics simulations, revealed significant microsecond-scale dynamics throughout the enzyme's water network, as detailed in our findings (Singh et al. 2019). Interestingly, introducing an inhibitor to this system slowed down these dynamics and disturbed the network to some extent (Vasa et al. 2019). The dwell time of zinc-bound water in the enzyme's pocket, as inferred from earlier magnetic relaxation dispersion studies (Denisov et al. 1999), matched the timing of these dynamic movements, underscoring their importance in catalytic efficiency (Vasa et al. 2018, 2019; Singh et al. 2020).
Current study explores the restructuring of the enzyme's water pocket structure and its consequences for the catalytic function of hCAII. We are specifically focused on the conformational changes that occur during the critical proton transport process in hCAII, examining if the movement of excess protons correlates with conformational changes in the protein and the water molecules in the Zn2+-pocket, as suggested by Taraphder et al. (2016). The mechanism by which enzymes promote proton transfer via dynamic networks of hydrogen bonds between amino acid residues and water molecules is well-documented (Nagle and Morowitz, 1978; Nagle and Tristram-Nagle 1983). The critical function of His64 in facilitating proton movement, acting mainly as a donor or acceptor, is highlighted by the significant decrease in proton transfer when His64 is replaced with alanine (Tu et al. 1989). Alterations in Trp5, Tyr7, Asn62, and Gln92 have also been observed to impact the enzyme's catalytic performance (Tu et al. 2002; Elder et al. 2004; Paul and Taraphder 2015). Neutron imaging suggests that a network of hydrogen-bonded water molecules within hCAII's active site links the zinc-associated water to the internal configuration of His64 (Roy and Taraphder, 2007; Maupin and Voth 2010). Studies using molecular dynamics have shown that when His64 faces inward, a chain of 3–4 water molecules is sufficient for effective proton transfer, whereas networks of 5–6 water molecules can facilitate even greater proton movement (Roy and Taraphder 2006; Maupin et al. 2008; Maupin and Voth 2010; Mikulski and Silverman 2010). The configuration of these water molecule clusters and the positioning of His64 are crucial for the enzyme's catalytic effectiveness (An et al. 2002; Maupin and Voth 2010). On the other hand, the Ser2 residue is thought to be involved in the entry of water into the Zn2+-binding pocket. Therefore, in view of this significant role of H64 in the function of hCAII and that of S2 as a gate-keeper for water, we commence by detailing the near-complete sequence-specific resonance assignments for the Ser2Ala/His64Ala variant of hCAII. This foundational work is poised to pave the way for subsequent explorations into the complex nature and arrangement of the water-protein network within hCAII. Essentially, our study aims to shed light on the elaborate connectivity of water molecules within the enzyme's active site, revealing the dynamic interactions that drive its catalytic efficiency.