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 16 s(-1). Our data suggest that HG binds to the apo-enzyme and that the apo-HGD.HG complex does not bind Fe(II) and dissociates slowly at approximately 0.028 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 holo-enzyme reduces molecular oxygen at a rate of 1.35x10(3) M(-1) s(-1) at 4 degrees C. 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.
Amaya
Alphonso A
AA
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211-3029, USA.
Brzezinski
Kotanee T
KT
Farrington
Natasha
N
Moran
Graham R
GR
D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D050560:Homogentisate 1,2-Dioxygenase; D006713:Homogentisic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D020035:Holoenzymes; D010105:Oxygenases; D049308:Dioxygenases; D050560:Homogentisate 1,2-Dioxygenase; D006713:Homogentisic Acid; D010100:Oxygen