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Amino-(3,4-dihydroxyphenyl)methyl phosphonic acid, the phosphonic analog of 3,4-dihydroxyphenylglycine, had been previously reported as a potent inhibitor of tyrosinase. The mechanism of the apparent enzyme inhibition by this compound has now been established. Amino-(3,4-dihydroxyphenyl)methyl phosphonic acid turned out to be a substrate and was oxidized to o-quinone, which evolved to a final product identified as 3,4-dihydroxybenzaldehyde, the same as for 3,4-dihydroxyphenylglycine. Monohydroxylated compounds (amino-(3-hydroxyphenyl)methyl phosphonic acid and amino-(4-hydroxyphenyl)methyl phosphonic acid) were not oxidized, neither was 4-hydroxy-L-phenylglycine. However, the relatively high K m for amino-(3,4-dihydroxyphenyl)methyl phosphonic acid (0.52 mM) indicated that competitive inhibition could not entirely explain the previously reported strong inhibitory effect (K i ¼ 50 and 97 lM for tyrosine and 3-(3,4-dihydroxyphenyl)alanine (Dopa) as substrates, respectively). Neither was the enzyme covalently inactivated to a significant degree. Spectroscopic and electrochemical analysis of the oxidation of a mixture of Dopa and the inhibitor demonstrated that the phosphonic compound reduced dopaquinone back to Dopa, thus diminishing and delaying the formation of dopachrome. This produces an apparent strong inhibitory effect when the reaction is monitored spectrophotometrically at 475 nm. In this peculiar case Dopa acts as a redox shuttle mediating the oxidation of the shorter phosphonic homolog. Decomposition of the phosphonic o-quinone to 3,4-dihydroxybenzaldehyde drives the reaction against the slightly unfavorable difference in redox potentials.Keywords: tyrosinase; redox exchange; quinone; phosphonic amino acids; 3,4-dihydroxybenzaldehyde.Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme widely distributed in nature. It catalyses the hydroxylation of monophenols to o-diphenols and the oxidation of the latter to o-quinones using molecular oxygen. Its action on the physiological substrate, L-tyrosine, produces L-3-(3,4-dihydroxyphenyl)alanine (L-Dopa) and then dopaquinone, which undergoes a series of nonenzymatic reactions leading to melanins [1]. The enzyme is responsible for melanization in animals and browning in plants. As browning in food products is an undesirable process, there has been a constant need in food industry for compounds preventing this reaction. Inhibition of mammalian tyrosinase has also been indicated as a possible approach to control human melanoma [2]. Although a large number of tyrosinase inhibitors have been described in the literature [3], the search for new natural products and synthetic compounds with such activity still continues [4]. Some of the most potent, competitive inhibitors include mimosine [5,6], tropolone [7,8], and kojic acid [9][10][11][12]. Some of us had previously shown that amino-(3,4-dihydroxyphenyl)methyl phosphonic acid, the phosphonic analog of 3,4-dihydroxyphenylglycine, was also a potent inhibitor of tyrosinase [13]. With a K i of 50 and 97 lM for tyrosin...

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A simple method is presented for the consecutive determination of protonation constants of polyprotic acids based on their formation curves. The procedure is based on generally known equations that describe dissociation equilibria. It has been demonstrated through simulation that the values obtained through the proposed method are sufficiently consistent with the actual values. In contrast with the universally known and applied Bjerrum’s method, no differences in the accuracy of determination of subsequent protonation constant values are observed. The proposed method requires the value of one of the protonation constants (e.g., of the first one, K 1) of the polyprotic acid. An iterative method is proposed for finding the value, starting from approximate values determined from the environment of point n̅ = 0.5(n̅-average degree of protonation). Apart from high accuracy, the proposed method also has didactic advantages because it illustrates the problem of multiple acid equilibria in a visual (almost graphic) manner. The proposed method may also provide initial constant values for numerical methods of nonlinear approximation of the formation curve.

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