Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

Abstract

Arginine methylation is a prevalent post‐translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N‐methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη‐hydroxyarginine generally demonstrated substrate inhibition and had improved KM values, which evoked a possible role in inhibitor design. We also revealed that the perceived degree of enzyme processivity is a function of both cofactor and enzyme concentration, suggesting that previous conclusions about PRMT sequential methyl transfer mechanisms require reassessment. Finally, we demonstrated a sequential ordered Bi–Bi kinetic mechanism for PRMT1, based on steady‐state kinetic analysis. Together, our data indicate a PRMT1 mechanism of action and processivity that might also extend to other functionally and structurally conserved PRMTs.

Mechanisms and Inhibitors of Histone Arginine Methylation

“…This may explain why certain steady-state kinetic studies showed that PRMT catalysis follows an ordered sequential mechanism. [27f,g,29] Together, we believe that our transient kinetic model provides a mechanistic explanation that unifies the seemingly contradictory steady-state kinetic studies reported in the literature.…”

“…On the other hand, when low‐affinity peptide substrates are used, the contribution of enzyme‐substrate binding (i. e., E‐H4 formation) would be negligible and can be ignored in the kinetic scheme, in which case the steady‐state kinetics would behave like a standard ordered sequential mechanism. This may explain why certain steady‐state kinetic studies showed that PRMT catalysis follows an ordered sequential mechanism ,,. Together, we believe that our transient kinetic model provides a mechanistic explanation that unifies the seemingly contradictory steady‐state kinetic studies reported in the literature.…”

“…On the other hand, steady-state kinetic studies performed by others support that an ordered sequential kinetic mechanism for PRMT1, [29] PRMT2, [27f] and PRMT6. [27g] In this model, SAM binds the enzyme active pocket first, followed by substrate binding and, after the methyl transfer, the methylated arginine product is the first to dissociate from the enzyme followed by SAH release.…”

TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma

“…al. for PRMT1 [ 21 ]. We found that switching the terminal aminomethyl (TP-064) to a methoxy moiety to obtain N-((2-(1-(2-methoxyethyl)piperidin-4-yl)pyridin-4-yl)methyl)-N-methyl-3-phenoxybenzamide (TP-064N) clearly reduced the inhibitory activity against PRMT4 (IC 50 2.5 ± 0.6 μM; Figure 1B ).…”

Independent transcriptomic and proteomic regulation by type I and II protein arginine methyltransferases

“…PRMT5 is the predominant type II enzyme ( Stopa et al., 2015 ). Importantly, while PRMT1 and PRMT9 have been shown to exhibit semi-processivity ( Brown et al., 2018 ; Gui et al., 2013 ; Jain et al., 2016 ; Osborne et al., 2007 ), PRMT enzymes are generally distributive ( Burgos et al., 2015 ; Hu et al., 2016 ; Jacques et al., 2016 ; Lakowski and Frankel, 2008 , 2009 ; Obianyo et al., 2008 ; Wang et al., 2013 , 2014 ) and are capable of scavenging each other's substrates ( Dhar et al., 2013 ). These observations suggest a complex enzyme/substrate interplay and imply potentially complementary cellular roles.…”