Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline

Abstract

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT.

Figure 1

Structures of PNMT inhibitors described in the text.

Figure 2

Active site of human PNMT (hPNMT) highlighting interactions with SK&F 29661 (2). Data from PDB ID 1HNN.

Figure 3

Reaction time course for PNMT activity in the absence (●) and presence of 100 nM (○), 200 nM (▼;)and 500 nM (▽) 3. After appropriate time intervals the reaction was quenched and the extent of product formation was measured.

Figure 4

Irreversible inactivation of hPNMT with 3. hPNMT (1 μM) was incubated in the absence of inhibitor (■), with 2.5 μM 2 (▽), or with 2.5 μM 3 (●). At the indicated times aliquots were diluted 1:50 into the standard assay mixture and the PNMT activity determined.

Figure 5

Stoichiometry of inactivation of hPNMT

Figure 6

Multiply charged mass spectrum of hPNMT labeled with 3. The inset shows the Maxent deconvolution. The theoretical M_r are 30724 Da and 30914 Da for unlabeled and labeled hPNMT, respectively.