Homogentisate 1,2-Dioxygenase (HGD; E.C. 22.214.171.124) cleaves homogentisate (HG) to maleylacetoacetate (MAA). A deficiency of HGD in mammals results in Alkaptonuria, a disease characterized by the production of a polymer of the quinone oxidation product of homogentisate and its accumulation in collagenous and cartilaginous tissues. The result of this is the development of arthritic symptoms.
In our lab, human HGD has been sub-cloned and HGD from P. aeruginosa cloned. Both express to give active enzyme in the E. coli cell. The aliphatic products of homogentisate and homogentisate analogs typically absorb strongly in the long wavelength edge of the UV spectral transitions (ca 300-350 nm, 10-15 mM-1cm-1), the absorbance of the substrates, intermediates and products will be used as a spectrophotometric reporter, providing kinetic evidence for individual chemical steps. Initial stopped flow studies will be concerned with investigate if such analogs modulate the relative rates of catalytic steps to favor accumulation of intermediates.
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