Roles of active site residues in catalysis, substrate binding, cooperativity and the reaction mechanism of the quinoprotein glycine oxidase.

摘要: The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward binding. It is good model for studying kinetics cooperativity, specifically being able separate those aspects protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, His-767. Structures glycine-soaked crystals obtained each. Different had differential effects on kcat K0.5 catalysis, substrate binding, Hill coefficients describing steady-state or Phe-316 Tyr-766 variants retained catalytic activity, albeit altered cooperativity. Substitutions His-583 revealed that it essential structure H583C PlGoxA no present crystals. H767A previously undetected reaction intermediate, carbinolamine product-reduced CTQ adduct, exhibited only negligible activity. results these experiments, as well native previous variants, enabled construction detailed mechanism reductive half-reaction oxidation. proposed includes three discrete intermediates are covalently bound during reaction, two which have now been structurally characterized by X-ray crystallography.