TLP hydrolytic activity in the presence of divalent metal ions.

The production and hydrolysis of 5-HIU was measured at 312 nm and is shown over a 10 minute period. Enzyme reactions were performed as described in the text. Briefly, 0.04 U uricase was equilibrated in 50 mM potassium phosphate buffer, pH 7.8 in the presence or absence of 100 µM divalent metal ions (Cu²⁺; results for other divalent metal cations not shown). Enzyme reactions were initiated with the addition of 100 µM freshly diluted uric acid (at time 0). Reactions were performed at 22°C in a Biorad spectrophotometer. Open squares (□): production of 5-HIU in the absence of metal ions. Closed squares (▪): production of 5-HIU in the presence of Cu²⁺. After approximately 3 minutes, the amount of 5-HIU peaked and underwent slow, spontaneous decomposition. Open circles (○): addition of 5.2 nM recombinant S. Typhimurium TLP at time 2.5 minutes in the absence of metal ions resulted in rapid hydrolysis of 5-HIU. Closed circles (•): in the presence of Cu²⁺ no hydrolysis occurred following addition of TLP. (Data for other metal ions not shown).