Purification and characterization of sepiapterin reductase from rat erythrocytes.

Sepiapterin reductase from rat erythrocyte hemolysate was purified 2000-fold to apparent homogeneity with 30% yield. The specific activity of the purified enzyme was 18 units/mg protein, and its molecular weight was 55 000. The enzyme consists of two identical subunits, each of which has a molecular weight of 27 500. The enzyme showed a single peak by isoelectric focusing with a pI of 4.9 and partial specific volume of 0.73 cm3/g. The amino acid composition was determined. pH optimum of the enzyme was 5.5. The equilibrium constant of 2.2.10(9) of the enzyme showed that the equilibrium lies much in favor of dihydrobiopterin formation from sepiapterin in rat erythrocytes. From steady-state kinetic measurements, ordered bi-bi mechanism was proposed to the reaction of sepiapterin reductase in which NADPH binds to free enzyme and sepiapterin binds next. NADP+ is released after the release of dihydrobiopterin. The Km values for sepiapterin and NADPH were 15.4 microM and 1.7 microM, respectively, and the Vmax value was 21.7 mumol/min per mg.