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

Kyle J Mamounis,Erik T Yukl,Victor L Davidson

doi:10.1074/jbc.ra120.013198

Abstract

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

Highlights

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia
PlGoxA is a robust tryptophylquinone protein, which allows for more detailed structure–function studies of active-site residues than has been possible previously with other CTQ- and TTQ-dependent enzymes
Mutation of the active-site Asp residue that corresponds to Asp-678 in the TTQ-dependent methylamine dehydrogenase [16] and the CTQ-dependent MmLodA [14] and MmGoxA [12] prevented the formation of the quinone cofactor

Summary

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It was shown that mutation of Asp-678 to Glu in the active site of PlGoxA eliminated the observed cooperativity of steady-state kinetic behavior, but it did not diminish the observed cooperativity of glycine binding [13] The explanation for this was that the mutation slowed the rate of one or more reaction steps such that binding of glycine was no longer even partially rate-limiting in the overall reaction. Residue His-583 of PlGoxA is seen in the crystal structure to interact with the glycine–CTQ adduct in the active site This suggests a possible role in substrate binding or catalysis or both. In this study, these four amino acid residues were altered by site-directed mutagenesis, and the effects of the mutations on glycine binding, glycine oxidation kinetics, and protein structure were determined. Alteration of the binding and kinetic mechanism by a H767A mutation allowed structural characterization of another previously undetectable reaction intermediate

Results

Discussion

Experimental procedures