Abstract
The interactions of Aspergillus flavus uricase with the substrates O2 and urate and the inhibitors xanthine, cyanide, periodate and hydroxylamine were investigated. Under equilibrium conditions O2 does not bind directly to the enzyme, and the absence of O2 had no effect on either the binding stoicheiometry or binding constant for xanthine, as measured by equilibrium dialysis and microcalorimetry. Cyanide, periodate and hydroxylamine inhibit uricase in a non-classical manner. A decrease in initial velocity to a steady-state inhibited velocity can be observed on a time scale of minutes. The time-dependence, which is unaltered by prior incubation with the inhibitors, is consistent with a first-order transition. Rate constants for induction of inhibition are linearly dependent on inhibitor concentration, but independent of urate and O2 concentrations. Radioactively labelled urate forms a stable but reversible complex with uricase in the presence of cyanide and O2. These results were used to deduce the nature of enzyme-bound intermediates and thus for the proposal of a novel mechanism for cyanide inhibition.
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Selected References
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- BAUM H., HUBSCHER G., MAHLER H. R. Studies on uricase. II. The enzyme-substrate complex. Biochim Biophys Acta. 1956 Dec;22(3):514–527. doi: 10.1016/0006-3002(56)90062-2. [DOI] [PubMed] [Google Scholar]
- BENTLEY R., NEUBERGER A. The mechanism of the action of uricase. Biochem J. 1952 Dec;52(4):694–699. doi: 10.1042/bj0520694. [DOI] [PMC free article] [PubMed] [Google Scholar]
- CANELLAKIS E. S., COHEN P. P. The end-products and intermediates of uric acid oxidation by uricase. J Biol Chem. 1955 Mar;213(1):385–395. [PubMed] [Google Scholar]
- Colman R. F., Chu R. The role of sulfhydryl groups in the catalytic function of isocitrate dehydrogenase. 3. Effect of N-ethylmaleimide on chemical and physical properties. J Biol Chem. 1970 Feb 10;245(3):608–615. [PubMed] [Google Scholar]
- Conley T. G., Priest D. G. Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase. Biochem J. 1980 Jun 1;187(3):727–732. doi: 10.1042/bj1870727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HUBSCHER G., BAUM H., MAHLER H. R. Studies on uricase. IV. The nature and composition of some stable reaction products. Biochim Biophys Acta. 1957 Jan;23(1):43–53. doi: 10.1016/0006-3002(57)90283-4. [DOI] [PubMed] [Google Scholar]
- MAHLER H. R., HUBSCHER G., BAUM R. Studies on uricase. I. Preparation, purification, and properties of a cuproprotein. J Biol Chem. 1955 Oct;216(2):625–641. [PubMed] [Google Scholar]
- Pitts O. M., Priest D. G. A steady-state kinetic investigation of the uricase reaction mechanism. Arch Biochem Biophys. 1974 Jul;163(1):359–366. doi: 10.1016/0003-9861(74)90487-1. [DOI] [PubMed] [Google Scholar]
- Pitts O. M., Priest D. G. Uricase reaction intermediate. Mechanism of borate and hydroxide ion catalysis. Biochemistry. 1973 Mar 27;12(7):1358–1363. doi: 10.1021/bi00731a016. [DOI] [PubMed] [Google Scholar]
- Priest D. G., Fisher J. R. Substrate activation with a xanthine oxidase reaction. An alternative to dismutation as an explanation in the chicken liver system. Eur J Biochem. 1969 Oct;10(3):439–444. doi: 10.1111/j.1432-1033.1969.tb00708.x. [DOI] [PubMed] [Google Scholar]
- Priest D. G., Pitts O. M. Reaction intermediate effects on the spectrophotometric uricase assay. Anal Biochem. 1972 Nov;50(1):195–205. doi: 10.1016/0003-2697(72)90499-x. [DOI] [PubMed] [Google Scholar]
- Truszkowski R. Uricase and its action: Preparation and properties of ox-kidney uricase. Biochem J. 1930;24(5):1359–1370. doi: 10.1042/bj0241359. [DOI] [PMC free article] [PubMed] [Google Scholar]