Probing mechanistic questions in the PLP- and O-dependent l-Arg oxidases.

The pyridoxal-5'-phosphate-dependent l-Arg oxidases are unusual in that they are able to catalyze 4-electron oxidations of arginine using only the PLP cofactor. No metals or other accessory cosubstrates are involved; only arginine, dioxygen, and PLP. The catalytic cycles of these enzymes are replete with colored intermediates whose accumulation and decay can be monitored spectrophotometrically. This makes the l-Arg oxidases excellent subjects for detailed mechanistic investigations. They are worth studying, because they can teach us much about how PLP-dependent enzymes modulate the cofactor (structure-function-dynamics) and how new activities can arise from existing enzyme scaffolds. Herein we describe a series of experiments that can be used to probe the mechanisms of l-Arg oxidases. These methods by no means originated in our lab but were learned from talented researchers in other enzyme fields (flavoenzymes and Fe(II)-dependent oxygenases) and have been adapted to fit the requirements of our system. We present practical information for expressing and purifying the l-Arg oxidases, protocols for running stopped-flow experiments to examine the reactions with l-Arg and with dioxygen, and a tandem mass spectrometry-based quench-flow assay to follow the accumulation of the products of the hydroxylating l-Arg oxidases.