Abstract
The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O(2) free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O(2) migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k(cat)/K(m) (O(2)), supporting multiple dynamic pathways for molecular oxygen to reach the active site.
Highlights
Hansenula polymorpha3 amine oxidase is the eukaryotic CAO that has been kinetically characterized in the most detail [2,3,4,5,6,7]
Implicit ligand sampling provides a complete three-dimensional map for the migration of O2 between favorable regions. This map is created by contouring energy isosurfaces representing elevated potential of mean force (PMF) values (1.8 and 3.0 kcal/mol, Fig. 4)
In HPAO, we identify two major regions that contain the most probable pathways from protein solvent boundary to the buried active site according to the implicit ligand sampling calculation (Fig. 4b)
Summary
HPAO Purification—Wild-type HPAO (wtHPAO) for crystallization was heterologously expressed in Saccharomyces cerevisiae and purified as described previously [7, 36] with modifications. An additional lower resolution data set was collected at ϭ 1.72 Å to optimally collect anomalous scattering associated with the bound xenon (for Xe fЉ ϭ 9.0 electrons) (supplemental Table S1). The ionic strength of all buffers was kept constant at 0.3 M by the addition of an appropriate amount of KCl. Data were fit directly to the Michaelis-Menten equation, and kcat was calculated using the active protein concentration as determined by phenylhydrazine titration. The trajectories were used as input for an implicit ligand sampling analysis [44] contained in the VMD software package [45], resulting in detailed three-dimensional free energy profiles for O2 placement inside the protein based on the assumption that the presence of gas molecules can be TABLE 1 Data collection and refinement statistics
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