Substrate activation in the urate oxidase reaction
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The enzyme urate oxidase (UO) is involved in nitrogen metabolism of many organisms. Urate oxidation is carried out by UO without the aid of a metal ion or cofactor. A catalytic diad has previously been proposed to be involved in formation of dianionic urate during the UO reaction. Proton inventory, Raman spectroscopy, and kinetic isotope effects (KIE) methods have been utilized to further investigate how UO accomplishes activation of urate for oxidation. A proton inventory was conducted to determine the number of protons in flight during each round of catalysis. A "bowl shaped" proton inventory at pH 8.0 is indicative of more than one proton being in flight during catalysis. This observation supports the proposed Thr-Lys catalytic diad being involved in dianion formation. Raman spectroscopy has been used along with the competitive inhibitor 8-nitroxanthine to show evidence for dianion formation upon the binding of substrate to UO. To further study how UO activates the substrate, new methods for measuring kinetic isotope effects (KIE) are being developed. The use of continuous flow mass spectrometry to measure carbon isotope effects (IE) at positions C4 and C5 of urate will give insight into the transition state structure of the reaction. Continuous flow MS has the advantage of measuring the naturally abundant isotopic ratios of solid samples. Another new technique is the use of isotope depleted samples to measure carbon IE's of multi-carbon molecules. These new techniques have the potential to be employed in the study of many enzymatic reactions.