Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase.
HGO (homogentisate 1,2-dioxygenase; EC 1.13.11.5) catalyses the O2-dependent cleavage of HGA (homogentisate) to maleylacetoacetate in the catabolism of tyrosine. Anaerobic purification of heterologously expressed Fe(II)-containing human HGO yielded an enzyme preparation with a specific activity of 28.3+/- 0.6 micromol x min(-1) x mg(-1) (20 mM Mes, 80 mM NaCl, pH 6.2, 25 degrees C), which is almost twice that of the most active preparation described to date. Moreover, the addition of reducing agents or other additives did not increase the specific activity, in contrast with previous reports. The apparent specificity of HGO for HGA was highest at pH 6.2 and the steady-state cleavage of HGA fit a compulsory-order ternary-complex mechanism (K(m) value of 28.6+/-6.2 microM for HGA, K(m) value of 1240+/-160 microM for O2). Free HGO was subject to inactivation in the presence of O2 and during the steady-state cleavage of HGA. Both cases involved the oxidation of the active site Fe(II). 3-Cl HGA, a potential inhibitor of HGO, and its isosteric analogue, 3-Me HGO, were synthesized. At saturating substrate concentrations, HGO cleaved 3-Me and 3-Cl HGA 10 and 100 times slower than HGA respectively. The apparent specificity of HGO for HGA was approx. two orders of magnitude higher than for either 3-Me or 3-Cl HGA. Interestingly, 3-Cl HGA inactivated HGO only twice as rapidly as HGA. This contrasts with what has been observed in mechanistically related dioxygenases, which are rapidly inactivated by chlorinated substrate analogues, such as 3-hydroxyanthranilate dioxygenase by 4-Cl 3-hydroxyanthranilate.
Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd, Vancouver, BC, Canada V6T 1Z3.
Edwin J A
EJ
Veldhuizen
Frédéric H
FH
Vaillancourt
Cheryl J
CJ
Whiting
Marvin M-Y
MM
Hsiao
Geneviève
G
Gingras
Yufang
Y
Xiao
Robert M
RM
Tanguay
John
J
Boukouvalas
Lindsay D
LD
Eltis
D000693:Anaerobiosis; D049308:Dioxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D048168:Escherichia coli K12 / Q000201:enzymology / Q000235:genetics; D050560:Homogentisate 1,2-Dioxygenase; D006713:Homogentisic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008298:Maleates / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013379:Substrate Specificity; D014162:Transfection / Q000379:methods
D004791:Enzyme Inhibitors; D008298:Maleates; D011994:Recombinant Proteins; C011905:maleoylacetic acid; D049308:Dioxygenases; D050560:Homogentisate 1,2-Dioxygenase; D006713:Homogentisic Acid; D010100:Oxygen