Table 1.

Kinetics of recombinant VT- and NV-AANAT

SubstratesKM (mM)*kcat (s−1)*Ki (mM)*kcat/KM (×103⋅s−1⋅mol−1)
Mean95% CI
+
VT-AANAT P. marinus
 Phenylethylamines
  PEA2.35 ± 2.054.77 ± 2.2230.5 ± 43.22.0311.341.17
  Tyramine1.44 ± 0.675.64 ± 1.2925.4 ± 17.33.923.461.43
 Indoleethylamines
  Tryptamine0.11 ± 0.073.75 ± 0.5842.8 ± 41.533.454.413.4
  Serotonin0.54 ± 0.444.93 ± 2.253.03 ± 1.489.0528.484.98
  OMS0.05 ± 0.023.36 ± 0.339.63 ± 3.5467.759.522.1
 Alkylaminesn.a. (Putrescine, spermidine, butylamine)
VT-AANAT C. milii
 Phenylethylamines
  PEA3.62 ± 0.9733.73 ± 2.679.313.372.02
  Dopamine10.7 ± 1.4116.52 ± 0.861.550.240.19
  Tyramine1.32 ± 0.1139.09 ± 0.9529.570.510.5
 Indoleethylamines
  Tryptamine0.71 ± 0.0917.64 ± 0.66201 ± 7225.03.52.8
  Serotonin0.59 ± 0.1423.98 ± 0.940.662.041.86
  OMS0.58 ± 0.0821.52 ± 0.9792.5 ± 33.737.125.84.53
 Alkylaminesn.a. (Putrescine, spermidine, butylamine)
NV-AANAT C. milii
 Phenylethylamines
  PEA1.61 ± 0.890.026 ± 0.0040.0160.0190.006
  Dopaminen.a.
  Tyramine44.87 ± 13.70.20 ± 0.030.0040.00030.0003
 Indoleethylamines
  Tryptamine2.6 ± 0.730.71 ± 0.0050.0270.0110.006
  Serotonin59.46 ± 91.40.63 ± 0.894.62 ± 8.030.0110.0070.004
  OMS7.44 ± 1.660.2 ± 0.020.0270.0080.005
 Alkylamines
  Putrescine5.19 ± 1.430.5 ± 0.080.10.040.02
  Spermidine6.38 ± 2.630.39 ± 0.050.0610.0190.04
  Butylamine2.09 ± 1.250.026 ± 0.0040.0120.0170.005
  • The catalytic efficiency (kcat) for the expressed VT-AANAT enzyme from Agnathans and Chondrichthyes was higher for indolethylamines than phenylethylamines; neither acetylated alkylamines. The kcat values were generally lower for NV-AANAT than the corresponding values for VT-AANAT; kcat values for alkylamines were higher than for arylalkylamines. KM, kcat, and Ki values were inferred from figures as shown in SI Appendix, Fig. S1 using Prism6.01 (GraphPad software). CI was calculated using GraphPad Prism6 (KM and kcat) and Quickcalcs (http://www.graphpad.com/quickcalcs/) software. CI, confidence interval; n.a., no activity; OMS, O-methylserotonin; PEA, phenylethylamine.

  • * Mean ± CI.

  • Strong autoinhibition was observed with serotonin; accordingly, the KM, kcat, and kcat/KM values are based on product formation below 1 mM and the Ki value is based on product formation above this value.