N-ACETYL-L-TYROSINE (NAT) AS A SUBSTRATE FOR MUSHROOM TYROSINASE

N-acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N-acetyl dopa formed is oxidized by the enzyme to N-acetyl dopaquinon e (lambda(max) = 390 +/- 10 nm). H2O2 and NH2OH each shortened the lag period of NAT hydroxylation by the enzyme. H2O2 had an effect on the changes with time in the spectrum of product(s) formed and on the spec trum of the final product(s) obtained when NAT was hydroxylated by mus hroom tyrosinase, in a manner suggesting that H2O2 converts N-acetyl d opaquinone to a pink-violet product(s) (lambda(max) = 490 nm), whereas such a product(s) was not formed in the absence of H2O2. A pink-viole t product(s) (lambda(max) 490 +/- 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH2OH or para a mino benzoic acid (PABA), probably as a result of an interaction betwe en N-acetyl dopaquinone and NH2OH or PABA forming mono-or di-oximes. K ojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) inhibited effect ively the rate of NAT hydroxylation by mushroom tyrosinase in the abse nce or presence of H2O2. When NAT was oxidized by the enzyme in the ab sence of kojic acid, N-acetyl dopaquinone was formed at once and a sho ulder at 490-530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a pe ak at 320 +/- 10 nm, was detected, suggesting that N-acetyl dopaquinon e! oxidizes kojic acid to the yellow product(s). Maltol (3-hydroxy-2-m ethyl-4H-pyran-4-one), a gamma-pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroo m tyrosinase. The addition of maltol at the plateau phase of the react ion resulted in an immediate decline in absorbance at 100 nm, suggesti ng that. maltol conjugates with N-acetyl dopaquinone, yielding a produ ct(s) characterized by a lower extinction coefficient at 400 nm than t hat of N-acetyl dopaquinone alone. The final brown-red product(s) form ed when NAT was hydroxylated by mushroom tyrosinase was bleached in th e presence of ascorbic acid or H2O2.