Effect of tetrahydropteridines on the monophenolase and diphenolase activities of tyrosinase

Figures & data

Scheme I Suggested kinetic reaction mechanism of tyrosinase acting on monophenol and o-diphenol, with the non-enzymatic reactions corresponding to the evolution of o-quinone (Ros et al., 1994 [18]). Where M is monophenol, D is o-diphenol, E is enzyme tyrosinase, E_d is Desoxytyrosinase, E_m is Mettyrosinase, E_mM is Mettyrosinase monophenol binding complex, E_mD is Mettyrosinase o-diphenol binding complex, E_ox is Oxytyrosinase, E_oxM is Oxytyrosinase monophenol binding complex, E_ox-M is covalently bound oxytyrosinase monophenol, E_oxD is covalently bound oxytyrosinase o-diphenol, Q is o-quinone, DC is dopachrome, and RH₂ is reductant.

Figure 1 Spectrophotometric register of dopachrome accumulation versus action time of tyrosinase on L-tyrosine. Curve (a): [M]₀ = 0.36 mM and [E]₀ = 7 nM. Curve (b): same enzyme concentration as (a) and [M]₀ = 1 mM. Curve (c): same substrate concentration as (a) and [E]₀ = 14 nM.

Figure 2 Representation of the values of rate of tyrosinase acting on L-tyrosine (), and of the lag period with respect to enzyme concentration. [M]₀ = 0.25 mM. • Values of obtained in the true steady-state. ♦ Rate values obtained considering the increase in absorbance at a fixed time from beginning of reaction. ▪ Values of lag period.

Figure 3 Representation of the rate of tyrosinase acting on L-tyrosine (), and of the lag period (τ) with different substrate concentrations. [E]₀ = 45 nM, • Values of obtained in the true steady-state. ▴ Rate values obtained considering the increase in absorbance at a fixed time from beginning of reaction, three min. ▪ Values of lag period.

Figure 4 Action of reductants on monophenolase activity of tyrosinase. Effect of 6BH₄ and 6,7-di-CH₃BH₄. Effect of 6BH₄. [L-tyrosine]₀ = 0.9 mM, [E]₀ = 30 nM. The concentrations of 6BH₄ were (a) 0; (b) 80 μM; (c) 10 μM and (d) 25μM.

Figure 5 Effect of reductants on the diphenolase activity of tyrosinase. Effect of 6BH₄. Spectrophotometric registers of the accumulation of dopachrome in the action of tyrosinase on L-dopa. The experimental conditions were: The initial concentration of o-diphenol [D]₀ = 1 mM, [E]₀ = 15 nM. The values of [6BH₄]₀ were: (a) 0; (b) 10 μM; (c) 20 μM and (d) 60 μM.

Figure 6 Effect of reductants on the monophenolase activity of tyrosinase in the steady-state. The steady-state was reached rapidly adding a quantity of o-diphenol so that [D] = [D]_ss = R[M]₀ with R = /. [M]₀ = 0.9 mM, [E]₀ = 62 nM, [D]_ss = 45 μM. Effect of 6BH₄. The concentrations of 6BH₄ were: a) 0; (b) 50 μM; (c) 65 μM and (d) 75 μM, respectively. Inset: Effect of AH₂. The values of initial concentration of ascorbic acid [AH₂]₀ were (a) 0; (b) 50 μM and (c) 75 μM.

Ros JR, Rodríguez-López JN, García-Cánovas F. Tyrosinase: Kinetic analysis of the transient phase and the steady-state. Biochim Biophys Acta 1994; 1204: 33–42

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