KINETIC AND CATALYTIC CHARACTERIZATION OF URATE OXIDASE FROM CHLAMYDOMONAS-REINHARDTII

Urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from Chlamydomonas reinhardtii is a tetramer of M(r) = 124 000, composed of similar-sized subunits and containing four atoms of copper per enzyme molecule (J. M. Alamillo, J. Cardenas and M. Pineda, Biochim. Biophys. Acta, 1076 (1991) 203-208). The enzyme exhibits a strict specificity toward its substrates, oxygen and urate, and has optimal activity in the pH 8.5-9.5 range, as determined by the V(max)/K(m) ratio. Purified uricase obeys hyperbolic kinetics for urate but shows a sigmoidal response to oxygen (Hill's coefficient = 3.7), indicative of a positive homotropic cooperativity. Under steady-state conditions, sets of intersecting lines were found in the double-reciprocal plots, which indicates that uricase reaction proceeds through a ternary complex involving enzyme, urate and oxygen. Since each substrate bonded to the enzyme in the absence of the other, a random mechanism is proposed for the complex formation. Uricase activity was competitively inhibited by hypoxanthine, xanthine and their 8-aza derivatives, as well as by oxonate, allantoin and CO2. Hydrogen peroxide acted as an uncompetitive inhibitor, and 1- and 9-methylurate showed mixed inhibition. Dissociation constants calculated from the effect of pH on K(m) and V(max) indicate that two dissociable groups with pK values of about 7.6 and 10.0 are probably involved in both substrate binding and catalytic oxidation. Inhibition by the group-specific reagents diethylpyrocarbonate arid phenylglyoxal strongly suggests that those dissociable groups are histidyl and arginyl residues. From these data a picture emerges in which copper and dissociable groups on the enzyme interact with C = O and NH groups on urate.