The narrow substrate specificity of human tyrosine aminotransferase – the enzyme deficient in tyrosinemia type II

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Abstract

Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Iγ subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pKa of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pKa (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/α-ketoglutarate substrate pair, which gives a steady state kcat value of 83 s−1. In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the kcat/Km value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Iα and Iγ aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.

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