Cytochrome P450 OleT utilizes hydrogen peroxide (HO) to catalyze the decarboxylation or hydroxylation of fatty acid (FA) substrates. Both reactions are initiated through the abstraction of a substrate hydrogen atom by the high-valent iron-oxo intermediate known as Compound I. Here, we specifically probe the influence of substrate coordination on OleT reaction partitioning through the combined use of fluorescent and electron paramagnetic resonance (EPR)-active FA probes and mutagenesis of a structurally disordered F-G loop that is distal from the heme-iron active site. Both probes are efficiently metabolized by OleT and efficiently trigger the formation of Compound I. Transient fluorescence and EPR reveal a slow product release step, mediated by the F-G loop, that limits OleT turnover. A single-amino acid change or excision of the loop reveals that this region establishes critical interactions to anchor FA substrates in place. The stabilization afforded by the F-G loop is essential for regulating regiospecific C-H abstraction and allowing for efficient decarboxylation to occur. These results highlight a regulatory strategy whereby the fate of activated oxygen species can be controlled at distances far removed from the site of chemistry.
The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein–protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein–protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.