Date of Award
Doctor of Philosophy (PhD)
Holz, Richard C.
Nitrile hydratase (NHase, E.C.22.214.171.124) is a metalloenzyme that catalyzes the hydration of nitriles into their corresponding amide under ambient conditions. NHase enzymes contain either a non-heme Fe3+ ion or a non-corrin Co3+ ion in their active site. NHase enzymes have industrial applications as biocatalysts in the large-scale production of acrylamide and nicotinamide, though it’s catalytic and biochemical properties are not fully understood. This research project provides insight into the active site maturation process and catalytic mechanism of NHase using peptide model complexes, site-directed mutagenesis and synthesis of deuterated proteins.Insight into the sequential maturation of the NHase active site has been provided in chapter 2. An eight amino acid peptide mimic of the metal binding motif of PtNHase was prepared and the metal bound complexes were analyzed using optical, EPR and MCD spectroscopy. These metallopeptide mimics clearly revealed that the metal binds into the active site in a high spin divalent state, then the equitorial cysteine residues are oxidized and finally the metal ion is oxidized to the trivalent ion. In chapter 3, the catalytic role of the axial cysteine ligand was examined through site directed mutagenesis, optical spectroscopy, kinetic analysis and X-ray crystallography. The role of the activator protein and the metal ion in active site maturation process was examined by expressing NHase in the absence and presence of Co2+ and/or activator protein and then elucidated through X-ray crytallography and kinetic analysis. This investigation strongly supports the role of activator protein in metal ion insertion and the maturation of the active site in conjuction with the metal ion. The axial ligand has been shown to be necessary for catalysis and metal ion insertion. A neutron diffraction investigation into the protonation states of active site residues supports the fact that a large and well ordered protein crystal is key to full data set collection for neutron diffraction. PtNHase is stable in its perdeuterated and deuterated states and large crystals, that diffract X-ray to >1.9 Å can be obtained. The perdeuterated protein has 60% activity of the wildtype and is able to grow into large crystals.
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