QM/MM studies on the catalytic mechanism of Phenylethanolamine N-methyltransferase

Kinetic Isotope Effects and Transition State Structure for Human Phenylethanolamine N-Methyltransferase

“…Although the final TS for hPNMT matches the measured intrinsic KIEs (Table 1), it is important to note this model uses simplified reactant structures and does not include noncovalent contributions from enzyme active site. However, the loose character of the hPNMT TS is consistent with previous studies on the hPNMT reaction mechanism, 9,10 as well as the TS structures of NSD2 14 and DNMT1, 15 all of which utilized more complex computational model systems.…”

“…Recently, density functional theory (DFT) 9 and quantum mechanics/molecular mechanics (QM/MM) 10 methods have been used to investigate the hPNMT reaction mechanism. These studies suggest that hPNMT catalyzes the formation of epinephrine through an S N 2 transition state (TS) in which the methyl transfer step is rate-limiting.…”

“…22 These isotope effects are in agreement with previous studies that support methyl transfer as the rate-limiting step of the PNMT reaction. 9,10 DFT calculations for NSD2 14 and DNMT1 15 suggest that the primary 36 S KIE on methyltransferase reactions is proportional to the loss of bond order between the carbon of the transferring methyl (C Me ) and the sulfur of SAM at the TS. The 36 S KIE for hPNMT (1.014 ± 0.005) is smaller than those measured for NSD2 (1.018 ± 0.008) 14 and DNMT1 (1.019 ± 0.006), 15 suggesting that hPNMT TS structure may retain somewhat greater C Me –S bond order.…”

“…9 In a separate investigation, Liu et al modeled the hPNMT reaction using QM/MM calculations that included the full protein solvated in a 30 Å water sphere. 10 This more computationally intensive study considered three discrete TS structures for the hPNMT reaction: (1) a nonrate-limiting deprotonation of norepinephrine, (2) a rate-limiting methyl transfer step, and (3) a nonrate-limiting deprotonation of epinephrine. The authors report a more expanded TS structure for the methyl transfer step (S–C Me –N distance = 4.66 Å); 10 however, the early character (C Me –S = 2.19 Å, C Me –N = 2.47 Å) of this TS structure is consistent with the models reported by Himo and co-workers, 9 as well as our simplified TS model (Figure 2).…”

“…10 This more computationally intensive study considered three discrete TS structures for the hPNMT reaction: (1) a nonrate-limiting deprotonation of norepinephrine, (2) a rate-limiting methyl transfer step, and (3) a nonrate-limiting deprotonation of epinephrine. The authors report a more expanded TS structure for the methyl transfer step (S–C Me –N distance = 4.66 Å); 10 however, the early character (C Me –S = 2.19 Å, C Me –N = 2.47 Å) of this TS structure is consistent with the models reported by Himo and co-workers, 9 as well as our simplified TS model (Figure 2). Comparison of the hPNMT TS structure to those of NSD2 14 and DNMT1 15 reveals similar distances along the methyl donor–acceptor axis (Supporting Information Table S1).…”