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Electrostatic interactions and the pH-dependent stability and mechanism of BCX Joshi, Manish D.


The pH-dependent activity of Bacillus circulans xylanase (BCX) is determined by ionization states of the nucleophile (Glu78, pK[sub a] 4.6) and the acid/base catalyst (Glul72, pK[sub a] 6.7). Inspection of the BCX structure shows that Glu78 and Glul72 are in similar environments and surrounded by equivalent, conserved active-site residues. Hence, there are no obvious reasons as to why their pK[sub a]'s are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK[sub a]’s (measured by ¹³C-NMR and pH-dependent activity profiles) of these two catalytic carboxylic acids and give rise to their difference in ionization behaviour. Results indicate that all of the conserved active-site residues act conceitedly in establishing the pK[sub a]'s of Glu78 and Glul72, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen-bonds serve to lower the pK[sub a]'s of Glu78 and Glul72. In contrast, neighbouring acidic groups can either lower or raise the pK[sub a]'s of the catalytic glutamic acids, using direct hydrogen-bonding interactions or reverse-protonation mechanisms. Analysis of several BCX structures indicates that Glu78 is preferentially stabilized over Glul72 in part by stronger hydrogen-bonds donated by rigidly held residues. Furthermore, theoretical calculations show that Glu78 has a lower pKa than Glul72 due to more favourable local and global electrostatics. Mutating neighbouring residues within the active-site modifies the BCX pH-optimum, achieving greater than wildtype levels of activity in isolated cases. However, activity is usually compromised due to loss of important ground and/or transition-state interactions. The roles that specific acidic groups play in the pH-dependent stability of BCX were also assessed by measuring the pK[sub a]'s, using ¹³C-NMR, of all seven aspartates. The pK[sub a]’s of all aspartate residues are less than corresponding values observed with random coil polypeptides, indicating that their ionization contributes favourably to the stability of the folded enzyme. Residues that were most conserved, most buried and most perturbed in pKa contributed the most to the pH-dependent stability of the enzyme and were stabilized extensively by either hydrogen-bonds or salt bridges, implicating a role for electrostatics in the protein interior of family 11 xylanases.

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