Introduction of Urease
Urease, classified as urea amidohydrolase (E.C. 3.5.1.5), emerges as a vital nickel-dependent enzyme across various organisms, from plants and algae to fungi and prokaryotes. Its pivotal role in the nitrogen cycle involves catalyzing the rapid hydrolytic decomposition of urea, producing ammonia and carbamate. This enzymatic activity induces a significant increase in pH, impacting both human health and agriculture. Pathogenic microorganisms, including Helicobacter pylori, Staphylococcus aureus, and Mycobacterium tuberculosis, exploit urease for survival and virulence, emphasizing its relevance in infectious diseases.
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In environmental contexts, urease's presence in soils contributes to ammonia release, affecting soil fertilization efficiency and inducing plant damage. This ammonia release also leads to soil pH increase and the formation of airborne particulate matter, contributing to atmospheric pollution and potential health hazards. The enzyme's widespread implications have prompted extensive scientific exploration, focusing on structure-function relationships and catalytic mechanisms. Understanding the detailed reaction mechanism of urease is not only essential for unraveling the mysteries of nitrogen metabolism but also holds significance in various industrial and medical applications. Over the years, extensive research has been conducted to delineate the structure-based reaction mechanism of urease, leading to fascinating discoveries and a more comprehensive understanding of this vital enzyme.
Fig. 1 Enzymatic steps for the urea hydrolysis (Mazzei L., et al. 2020).
Crystal Structures of Ureases
The journey into deciphering the reaction mechanism of urease began with the determination of its crystal structures. The elucidation of the crystal structure of Klebsiella aerogenes urease (KAU) marked a significant milestone in 1995. Subsequent to this breakthrough, other ureases, including Sporosarcina pasteurii urease (SPU), were structurally characterized. These studies revealed a conserved di-nickel active site at the heart of urease's catalytic prowess.
The Active Site Architecture
The active site of urease, comprised of two nickel ions coordinated by a network of amino acid residues, serves as the epicenter for urea hydrolysis. The crystal structures of KAU and SPU unveiled a striking similarity in their tertiary and quaternary structures. The coordination environment of the nickel ions in the active site demonstrated the presence of key residues, such as αLys220*, αHis249, αHis275, αHis137, αHis139, and αAsp363, orchestrating the catalytic dance. The hydration environment surrounding the active site, including solvent molecules denoted as W(1), W(2), W(3), and W(B), played a crucial role in stabilizing the di-nickel cluster.
Spatiotemporal Dynamics of the Active Site
The dynamics of the active site emerged as a focal point of investigation. The crystal structure of native SPU, captured in an open conformation, showcased a mobile flap covering the active site. This elusive open state was significant for understanding urease's functioning. Concomitantly, studies on SPU bound to diamidophosphate (DAP), a transition state analog, revealed a closed conformation of the flap. This finding validated the hypothesis of a dynamic flap, capable of assuming open and closed states during the catalytic cycle.
The Bridging Hydroxide Mechanism
Inspired by these structural revelations, researchers proposed the "bridging hydroxide mechanism." According to this hypothesis, urea enters the active site when the flap is open, replacing solvent molecules. αHis222, forming a hydrogen-bonding network, stabilizes the initial binding of urea to the more electrophilic Ni(1). In a bidentate binding mode, urea chelates both nickel ions, activating the substrate for hydrolysis. The bridging hydroxide, symmetrically positioned between the nickel ions, acts as both the nucleophile attacking the carbonyl C of urea and the general acid in subsequent steps.
Controversies and Confirmations
The bridging hydroxide mechanism faced skepticism initially, particularly regarding the kinetic feasibility of a doubly coordinated nucleophile. However, subsequent studies, including the crystal structure of SPU bound to phosphate and a computational approach, provided support. The controversy heightened with discussions around the coordination state of urea during binding. Computational studies favored flap closure before binding of one amido-NH2 group to Ni(2), challenging the original hypothesis. However, the crystal structure of SPU bound to boric acid and subsequent studies on the mobile flap reaffirmed the bridging hydroxide mechanism.
The Active Site Flap's Role
The helix-turn-helix motif covering the active site cavity, featuring the conserved αHis320/αHis323 residue, became a central player. The mobile flap's ability to switch between open and closed conformations was experimentally proven, further emphasizing its role in stabilizing substrates or transition states. The flap's closure was deemed essential for catalysis, with cases of inactivated urease supporting this notion.
pH-Dependent Conformational Changes
An intriguing aspect of urease's catalytic cycle emerged with pH-dependent conformational changes. The pH of crystallization influenced the flap's stability in open or closed conformations. At higher pH, the flap was stabilized in the closed conformation, forming crucial interactions with αAsp224 and αArg339. Conversely, at lower pH, the flap tended to stay open, exposing the active site to solvent molecules.
Fluoride as a New Player
The bridging hydroxide hypothesis gained further support with studies on fluoride inhibition. Initial kinetic studies on KAU hinted at fluoride replacing the Ni-bridging solvent molecule. Subsequent structural investigations on SPU confirmed this, revealing two fluoride ions replacing the bridging solvent and the terminal water molecule on Ni(1). The fluoride inhibition mechanism was deemed consistent with the bridging hydroxide hypothesis.
Ternary Complex Structure
The quest for a deeper understanding culminated in the determination of a ternary complex structure of SPU with fluoride and urea. This groundbreaking structure showcased a fluoride ion occupying the bridging position, validating previous kinetic and structural findings. Crucially, urea was observed binding in a bidentate mode to Ni(1) and Ni(2), providing a definitive structural snapshot of the enzyme-substrate complex.
Conclusion
The structural exploration of urease's reaction mechanism has been a captivating odyssey, marked by controversies, confirmations, and paradigm shifts. From the initial crystal structures to the dynamic interplay of the active site flap, the intricate details have gradually unfolded. The bridging hydroxide mechanism, once met with skepticism, now stands on a solid foundation, supported by crystallography, kinetics, and computational studies. As the journey continues, new insights into the spatiotemporal dynamics of urease promise to unravel further intricacies, making this enzyme a perennial source of fascination for bioinorganic chemists.
References
- Mazzei L.; et al. The structure-based reaction mechanism of urease, a nickel dependent enzyme: Tale of a long debate. JBIC Journal of Biological Inorganic Chemistry. 2020, 25(6): 829-845.
- Mobley H L T. Urease. Helicobacter Pylori: Physiology and Genetics. 2001: 177-191.
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