Is Water Required for the Structure and Function of a ...



Is Water Required for the Structure and Function of a Protease Enzyme?Key words: Protein, enzyme, protease, hydrolysis, solvent-free proteinsEnzymes are proteins. A protease is an enzyme that catalyses the hydrolysis of other proteins. Proteases provide an alternative chemical pathway with lower activation energy for the breaking of peptide bonds between two joined amino acids with the addition of a water molecule (fig.1). This reaction occurs within the active site of a protease, meaning that once the active site is empty of products it can be used again, fulfilling the definition of a catalysis. Proteases are required in important biological processes such as digestion and cell signalling.Areas of application for this research are the use of proteases in very dry environments, e.g. desert areas and space. By removing the need for water to dissolve the enzyme (solvating water), many more water molecules can be used for hydrolysis, making the process thousands of times more water efficient. However, removing water from a protein causes serious problems with activity.center22778500Figure 1: a skeletal structure mechanism of polypeptide hydrolysis. Proteins are highly folded macro molecules (molecules containing very large numbers of atoms) formed from polypeptide chains comprised of amino acids (joined via a peptide link) that are often solvated within aqueous solutions. The sequence of amino acids is highly specific for each protein, with the order along the polypeptide chain forming the primary structure of a protein (fig.2a). Certain sections of the polypeptide chain can interact through hydrogen bonding between amide hydrogens and carbonyl oxygens, folding the chain and forming secondary structures such as α-helices and β-sheets (fig.2b). The interaction of these secondary structures with similar neighbouring structures, again through hydrogen bonding, leads to tertiary structure and the formation of the fully folded protein structure.453638-59960570031419800Figure 2: A. an example section of a primary sequence (Alanine, Lysine, Histidine, and Valine), B. cartoon representations of (a) α-helix and (b) β-sheet CITATION Nic20 \l 2057 [1]. For a protein to fold, the structure must be more energetically favourable when folded than when unfolded. The stabilisation of a protein’s structure relies on several effects including the intra-chain hydrogen bonding and the hydrophobic effect. The R-groups on several amino acid residues are hydrophobic (non-polar), and therefore it is unfavourable for water, a polar molecule, to interact with them. This effect is so large that most of these non-polar residues are ‘buried’ within the centre of a protein to remove the interactions. This leaves the hydrophilic (polar) residues, including -COOH, -OH, exposed on the exterior of the protein, which form hydrogen bonds with the many water molecules solvating the proteinCITATION Cam16 \l 2057 [2]. The hydrophobic effect is necessary for folding, therefore, if there is no water, there is no hydrophobic effect, and the protein will not fold.39592251651000Figure 3: Cartoon displaying the solvating water (hydration shell, red and white) surrounding a protein (blue) CITATION HFr09 \l 2057 [4].This means that the removal of all water molecules would cause the protein to deactivate for two reasons: water molecules are required for the catalytic activity, and solvation levels of water are required for the hydrophobic effect. However, for a protein to be solvated there must at minimum be a monolayer of water surrounding the protein. This is known as a hydration shell (fig.3). Any less water and the enzyme is not technically solvatedCITATION Cos97 \l 2057 [3]. For most proteins the hydration shell contains thousands of water molecules. Therefore, the lack of a full hydration shell doesn’t mean the removal of all water molecules surrounding a protein, some can remain associated and used during catalysis. If the surface interactions with water (hydrogen bonds) could be mimicked it would solve the problem with unfolding. This has recently been achieved through a two-step process: chemically charging the surface of the protein followed by the addition of oppositely charged surfactant molecules. Removing water molecules to below the level for solvation was done through a process called lyophilization; cooling molecules down to below the freezing point and placing them under a vacuum causes a phase transition from solid to gas. The protease is now in a solvent-free state, with experiments showing fewer than 30 water molecules per protein. The whole process was shown to have little detrimental effect on the structure of the protein, displayed here as small percentage changes to secondary structure (table 1). Note, as a protease catalyses the breakdown of proteins, the substrate protein also needs to be in a solvent-free state.Table 1: Secondary structure percentages of a protease before and after modification.Sampleα-Helix / %β-Sheet / %Other / %Protease103456Solvent-free protease93160559435-4699000Figure 4: Graph showing the decrease in secondary structure of the solvent-free substrate protein when mixed with the solvent-free protease. In recent research two modified solvent-free proteins (the protease and substrate) were mixed and the hydrolysis activity was tracked by studying changes in structure of the substrate. During the reaction it was shown that protein structure was altered meaning that peptide bonds were being broken by the protease and that the solvent-free protease remained active (fig.3). This technique could be applied to numerous industrially relevant proteases, allowing for their use in arid environments and potentially for use within space exploration, where it is challenging and expensive to find and transport large quantities of water.00Dr Jonathan Furze is the postdoctoral outreach assistant at the School of Chemistry, University of Bristol. During his PhD he focused upon designing a functional solvent-free liquid protease and developing a new technique to determine the activity. He is currently supporting all Bristol ChemLabS outreach-based activities at The University of Bristol. Is Water Required for the Structure and Function of a Protease Enzyme?QuestionsWhat does a protease do? [1 mark]In figure 1, what type of reaction is shown [1 mark]What are the R-Groups (Alanine, Lysine, Histidine, and Valine) shown in figure 2. (Draw or name). (4 marks)Why are hydrophilic (polar) amino acid R groups such as -COOH and -OH, exposed on the exterior of a water-soluble protein? [2 marks]What is a hydration shell? shown [1 mark]How can a protease be converted to a solvent free state? [2 marks]What is a peptide bond? [1 mark]Where on a protein molecule do enzymic reactions take place? [1 mark]AnswersIs Water Required for the Structure and Function of a Protease Enzyme?QuestionsWhat does a protease do? [1 mark]In figure 1, what type of reaction is shown [1 mark]What are the R-Groups (Alanine, Lysine, Histidine, and Valine) shown in figure 2. (Draw or name). (4 marks)Why are hydrophilic (polar) amino acid R groups such as -COOH and -OH, exposed on the exterior of a water-soluble protein? [2 marks]What is a hydration shell? shown [1 mark]How can a protease be converted to a solvent free state? [2 marks]What is a peptide bond? [1 mark]Where on a protein molecule do enzymic reactions take place? [1 mark] ................
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