Article ; Online: Probing mechanistic questions in the PLP- and O
2023 Volume 685, Page(s) 493–529
Abstract: 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 ...
| Abstract | 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. |
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| MeSH term(s) | Humans ; Oxidoreductases ; Pyridoxal Phosphate ; Oxygenases ; Arginine ; Oxygen |
| Chemical Substances | Oxidoreductases (EC 1.-) ; Pyridoxal Phosphate (5V5IOJ8338) ; Oxygenases (EC 1.13.-) ; Arginine (94ZLA3W45F) ; Oxygen (S88TT14065) |
| Language | English |
| Publishing date | 2023-04-26 |
| Publishing country | United States |
| Document type | Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. |
| ISSN | 1557-7988 |
| ISSN (online) | 1557-7988 |
| DOI | 10.1016/bs.mie.2023.03.010 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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