Evidence of the Participation of Remote Residues in the Catalytic Activity of Co-Type Nitrile Hydratase from Pseudomonas putida

摘要

Active sites may be regarded as layers of residues, whereby the residuesnthat interact directly with substrate also interact with residues in a second shell andnthese in turn interact with residues in a third shell. These residues in the second andnthird layers may have distinct roles in maintaining the essential chemical properties ofnthe first-shell catalytic residues, particularly their spatial arrangement relative to thensubstrate binding pocket, and their electrostatic and dynamic properties. The extent tonwhich these remote residues participate in catalysis and precisely how they affect first-nshell residues remains unexplored. To improve our understanding of the roles ofnsecond- and third-shell residues in catalysis, we used THEMATICS to identifynresidues in the second and third shells of the Co-type nitrile hydratase fromnPseudomonas putida (ppNHase) that may be important for catalysis. Five of thesenpredicted residues, and three additional, conserved residues that were not predicted,nhave been conservatively mutated, and their effects have been studied both kinetically and structurally. The eight residues have nondirect contact with the active site metal ion or bound substrate. These results demonstrate that three of the predicted second-shellnresidues (R-Asp164, β-Glu56, and β-His147) and one predicted third-shell residue (β-His71) have significant effects on the catalyticnefficiency of the enzyme. One of the predicted residues (R-Glu168) and the three residues not predicted (R-Arg170, R-Tyr171, andnβ-Tyr215) do not have any significant effects on the catalytic efficiency of the enzyme.