Enzyme kinetic and molecular modelling studies of sulphur-containing substrates of phenylalanine 4-monooxygenase

Figures & data

Table I.  Molecular modelling of human PAH active site.

	Distance of oxidation site from (Å)
Substrate	Fe^2+	H2O	BH4 (oxygen)
Phe	3.09^*	2.17	4.53	5.19
Met	3.69^*	3.89	6.36	6.24
SMC	4.92	4.40	6.98	6.65
SCMC	4.93	4.40	6.96	6.65
ERG	3.14^*	1.88	3.71	5.46

^* Distance < 4.2 Å is adequate for the introduction of molecular O₂ [18].

Figure 1.  (a) 3-D schematic diagram of Phe-PAH interactions. (b) 3-D schematic diagram of Met-PAH interactions. (c) 3-D schematic diagram of SMC-PAH interactions. (d) 3-D schematic diagram of SCMC-PAH interactions. (e) 3-D schematic diagram of SME-protein interactions. Molecular models of the predicted complexes formed between PAH and the substrates Phe (a), Met (b), SMC (c), SCMC (d) and SME (e). Atoms are colour coded as C: green, N: blue, O: red, Fe: pink, S: yellow. Potential hydrogen bonds are indicated by dashed red lines, and PAH residues in van de Waals contact with the ligands are boxed. All inter atomic distances are given in Å.

Table II.  Enzyme kinetic data for activated pooled rat cytosolic fraction PAH assays using Phe, Met, SME, SMC and SCMC as substrates.

Substrate	n	Km (mM)	Vmax (nmole min^− 1 mg protein^− 1)	ClE (μl min^− 1 mg protein^− 1)	Ki (mM)	Coupling efficiency
Phe	6	0.26 ± 0.08	3.72 ± 4.74	14.30 ± 0.72	ND	0.95 ± 0.05
Met	6	7.75 ± 3.01	63.88 ± 6.81	8.24 ± 0.41	8.11 ± 0.44	0.94 ± 0.06
SMC	6	43.25 ± 7.76	29.73 ± 3.79	0.69 ± 0.03	41.53 ± 2.49	0.94 ± 0.05
SCMC	6	14.73 ± 8.91	48.11 ± 10.69	3.26 ± 0.16	13.14 ± 0.66	0.95 ± 0.05
SME	6	0.45 ± 0.05	5.09 ± 1.34	11.31 ± 8.9	0.50 ± 0.04	ND

n = number of experiments. Each experiment was determined in duplicate with associated blanks. PAH was activated by pre-incubation with 1.0 mM lysophosphatidylcholine for 3.0 min at 37°C. Cl_E = V_max/K_m. Coupling efficiency = ratio of metabolite produced (Tyr, Met S-oxides, SMC S-oxides, SCMC S-oxides)/NADH oxidised using 2.0 mM phenylalanine/40.0 mM SCMC/50.0 mM SMC/20.0 mM Met and 100 μM BH₄. ND = not determined.

Table III.  Enzyme kinetic data for activated pooled human cytosolic fraction PAH assays using Phe, Met, SME, SMC and SCMC as substrates.

Substrate	n	Km (mM)	Vmax (nmole min^− 1 mg protein^− 1)	ClE (μl min^− 1 mg protein^− 1)	Ki (mM)	Coupling efficiency
Phe	6	0.64 ± 0.03	91.21 ± 5.47	142.51 ± 0.71	ND	0.96 ± 0.04
Met	6	3.10 ± 0.16	59.13 ± 2.95	18.59 ± 1.01	3.50 ± 0.18	0.95 ± 0.05
SME	6	0.30 ± 0.03	15.97 ± 1.25	53.31 ± 2.67	0.41 ± 0.03	ND
SMC	6	18.32 ± 1.00	31.28 ± 1.41	1.70 ± 0.09	17.32 ± 0.77	0.94 ± 0.05
SCMC	6	4.60 ± 0.23	50.26 ± 2.57	10.92 ± 0.16	5.01 ± 0.25	0.95 ± 0.05

n = number of experiments. Each experiment was determined in duplicate with associated blanks. PAH was activated by pre-incubation with 1.0 mM lysophosphatidylcholine for 3.0 min at 37°C. Cl_E = V_max/K_m. Coupling efficiency = ratio of metabolite produced (Tyr, Met S-oxides, SMC S-oxides, SCMC S-oxides)/NADH oxidised using 1.0 mM phenylalanine/20.0 mM SCMC/40.0 mM SMC/15.0 mM Met and 100 μM BH₄. ND = not determined.

OA Andersen, T Flatmark, and E Hough. (2002). J Mol Biol 320:1095–1108.

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