Two tyrosine residues activate phenolic substrates in vanillyl-alcohol oxidase

What is it about?

The crystal structure of VAO reveals that two tyrosine residues, Tyr-108 and Tyr-503, are positioned to facilitate this deprotonation. To investigate their role in catalysis, we created three VAO variants, Y108F, Y503F, and Y108F/Y503F, and studied their biochemical properties. Steady-state kinetics indicated that the presence of at least one of the tyrosine residues is essential for efficient catalysis by VAO. Stopped-flow kinetics revealed that the reduction of VAO by chavicol or vanillyl alcohol occurs at two different rates: kobs1, which corresponds to its reaction with the deprotonated form of the substrate, and kobs2, which corresponds to its reaction with the protonated form of the substrate. In Y108F, Y503F, and Y108F/Y503F, the relative contribution of kobs2 to the reduction is larger than in wild-type VAO, suggesting deprotonation is impaired in these variants. Binding studies disclosed that the competitive inhibitor isoeugenol is predominantly in its deprotonated form when bound to wild-type VAO, but predominantly in its protonated form when bound to the variants. These results indicate that Tyr-108 and Tyr-503 are responsible for the activation of substrates in VAO.

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Why is it important?

Studying the role of active site residues provides new insights into the catalytic mechanism of VAO and related enzymes that oxidize para-substituted phenols.

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