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Exploring molecular oxygen pathways in Hansenula polymorpha copper-containing amine oxidase.

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.

SUBMITTER: Johnson BJ

PROVIDER: S-EPMC3081669 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Exploring molecular oxygen pathways in Hansenula polymorpha copper-containing amine oxidase.

Johnson Bryan J BJ Cohen Jordi J Welford Richard W RW Pearson Arwen R AR Schulten Klaus K Klinman Judith P JP Wilmot Carrie M CM

The Journal of biological chemistry 20070404 24

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, ...[more]

PMID: 17409383

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Project description:The structural underpinnings of enzyme substrate specificity are investigated in a pair of copper amine oxidases (CAOs) from Hansenula polymorpha (HPAO-1 and HPAO-2). The X-ray crystal structure (to 2.0 A resolution) and steady state kinetic data of the second copper amine oxidase (HPAO-2) are presented for comparison to those of HPAO-1. Despite 34% sequence identity and superimposable active site residues implicated in catalysis, the enzymes vary considerably in their substrate entry channel. The previously studied CAO, HPAO-1, has a narrow substrate channel. In contrast, HPAO-2 has a wide funnel-shaped substrate channel, which also contains a side chamber. In addition, there are a number of amino acid changes within the channels of HPAO-2 and HPAO-1 that may sterically impact the ability of substrates to form covalent Schiff base catalytic intermediates and to initiate chemistry. These differences can partially explain the greatly different substrate specificities as characterized by k(cat)/K(m) value differences. In HPAO-1, the k(cat)/K(m) for methylamine is 330-fold greater than for benzylamine, whereas in HPAO-2, it is benzylamine that is the better substrate by 750-fold. In HPAO-2, an inflated (D)k(cat)/K(m)(methylamine) in relation to (D)k(cat)/K(m)(benzylamine) indicates that proton abstraction has been impeded more than substrate release. In HPAO-1, (D)k(cat)/K(m)(S) changes little with the slow substrate and indicates a similar increase in the energy barriers that control both substrate binding and subsequent catalysis. In neither case is k(cat)/K(m) for the second substrate, O(2), significantly altered. These results reinforce the modular nature of the active sites of CAOs and show that multiple factors contribute to substrate specificity and catalytic efficiency. In HPAO-1, the enzyme with the smaller substrate binding pocket, both initial substrate binding and proton loss are affected by an increase in substrate size, while in HPAO-2, the enzyme with the larger substrate binding pocket, the rate of proton loss is differentially affected when a phenyl substituent in the substrate is reduced to the size of a methyl group.

Dataset Information

Exploring molecular oxygen pathways in Hansenula polymorpha copper-containing amine oxidase.

Publications

Exploring molecular oxygen pathways in Hansenula polymorpha copper-containing amine oxidase.

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