Inhibition of Ring-Cleaving Dioxygenases by Natural Amino Acids

Inhibition of enzyme catalysis is critical for regulating cellular metabolism. Gentisate and salicylate 1,2-dioxygenases (GDO and SDO) are primary enzymes in the key gentisic pathway, which perform O₂-dependent degradation of aromatic compounds with the aid of a ferrous cofactor. Here, we report on the inhibition of the reaction catalysis of GDO and SDO by natural amino acids. Steady-state Michaelis-Menten studies monitoring the rate of the reaction in the presence of 18 amino acids show that l-histidine primarily inhibits SDO- and GDO-mediated catalysis. In addition, l-threonine and l-asparagine also inhibit the SDO-catalyzed chemical reaction with a diminished capacity. All amino acids competitively inhibit the catalysis suggesting their binding in the catalytic cavity. While l-histidine exhibits inhibition with a binding affinity of 80 μM (SDO) and 200 μM (GDO), d-histidine does not influence the rate of the chemical reaction, demonstrating that the chirality of the amino acid is critical for supporting optimal interactions that enable binding into the catalytic pocket. Lack of inhibition by small amino acids such as l-glycine suggests that the binding of the inhibitor is mediated by both the metal-ligating backbone carboxylate group and favorable interactions between the side chain and the catalytic cavity. Using nitric oxide as a surrogate for O₂ and EPR spectroscopy, we show that inhibitor binding does not obstruct the access of O₂ to the active site. In addition, EPR results demonstrate that unlike substrate binding, which introduces distortions, inhibitor binding does not alter the geometry of the metal center.