Reply to Frankel: Inconvenient Truths for PRMT6 Kinetic Studies

Abstract

This is a response to a letter by Frankel (1) Frankel questioned our assignment of a rapid equilibrium random kinetic mechanism for PRMT6 (2). Our mechanistic assignment is based on both product and dead-end inhibition experiments for four different peptide substrates. In contrast, Lakowski and Frankel used only product inhibition experiments with one peptide (3). The product inhibition data differ in the pattern afforded by the asymmetric dimethylarginine (ADMA)-containing peptides. They observed noncompetitive inhibition, whereas we saw competitive patterns in all four scenarios. Additionally, we saw no inhibition when S-adenosyl-l-methionine (SAM) was the varied substrate and ADMA-containing peptides were the product inhibitors at saturating amounts of substrate; an uncompetitive pattern is expected for their mechanism (3). Furthermore, an uncompetitive inhibition pattern, which is expected for an ordered mechanism, was not observed in our dead-end studies. A random mechanism is also supported by structural/biophysical studies. First, the IC50 values of C21, an irreversible inhibitor, are similar for PRMT6, regardless of whether C21 is preincubated with PRMT6 in the absence or presence of SAM (2). Second, our PRMT-targeted activity-based probes label PRMT6 in the absence of SAM (4). Because these probes are substrate analogs, these data indicate that SAM is not required for substrate/inhibitor binding (4). Finally, the N-terminal helices that divide and help form the SAM- and peptide/protein-binding pockets are flexible and are not always observed in structures, even in the presence of S-adenosyl-l-homocysteine (5, 6), which suggests that there is no structural requirement for SAM to bind prior to a peptide/protein substrate.