Spectrophotometric determination of homogentisate using Aspergillus nidulans homogentisate dioxygenase.

The presence of homogentisic acid (HGA) in urine is diagnostic for alkaptonuria, a classical example of a biochemical lesion resulting from a single gene trait. We describe here simple culture conditions which induce the synthesis of high levels of homogentisate dioxygenase activity in mycelia from the filamentous ascomycete Aspergillus nidulans. Crude enzyme preparations, showing an apparent Km of 9 microM for homogentisate and an optimal pH of 6.5-7.0 are rather stable and highly specific for homogentisate. Thus, the reaction is not competed by a large molar excess of a number of substrate structural analogues, including phenylacetate and its 2-, 3-, and 4-hydroxy derivatives, phenylalanine, tyrosine, phenylpyruvate, and gentisate. We demonstrate how this enzyme preparation can be used in sensitive, spectrophotometric enzymatic determination of this compound. The accuracy is almost indistinguishable from that obtained by HPLC. The method can be applied to routine determination of homogentisate in human urine. A 1-liter culture of the mold provides sufficient enzyme activity for 1500 enzymatic assays.