Kinetic analysis of human homogentisate 1,2-dioxygenase

Homogentisate 1,2-dioxygenase (HGD) is a mononuclear Fe(II)-dependent oxygenase that catalyzes the third step in the pathway for the catabolism of tyrosine, the conversion of homogentisate (HG) to maleylacetoacetate (MAA). We have heterologously expressed and purified native human HGD in the apo form. Steady-state analysis varying the concentration of both HG and molecular oxygen shows that the purified enzyme has a turnover number of 16s(-1). Our data suggest that HG binds to the apoenzyme and that the apo-HGD . HG complex does not bind Fe(II) and dissociates slowly at similar to0.028 s(-1). The rate constant for the dissociation of Fe(II) from the holo-enzyme as measured under anaerobic conditions is 0.00004 s(-1) and indicates that this process is not relevant in steady-state turnover. The addition of HG and molecular oxygen to the holo-enzyme is formally random as the holoenzyme reduces molecular oxygen at a rate of 1.35 x 10(3) M-1 s(-1) at 4 degreesC. The term ordered with respect to the addition of substrates is most descriptive as the rate of reduction of molecular oxygen must increase in the presence of HG to sustain the observed turnover number. (C) 2003 Elsevier Inc. All rights reserved.