| Abstract
| - Klebsiella aerogenes urease is a Ni-containing enzyme(two Ni per αβγ unit) that is purifiedas an apoprotein from cells grown in Ni-free medium. Partialactivation of urease and UreD-ureaseapoproteins is achieved in vitro by incubation in the presence ofNi(II) and CO2, whereas incubation ofthese proteins with Ni alone leads to the formation of inactive species[Park, I.-S., & Hausinger, R. P.(1995) Science 267, 1156−1158]. Here we determinedthe kinetics of these inhibitory reactions anddemonstrated the presence of two Ni ions per αβγ unit in theinactive proteins. Although metal-substitutedurease has never been purified from Ni-deprived cells, several othermetal ions were shown to bind to theurease apoproteins. Divalent Zn, Cu, Co, and Mn all inhibited Ni-and CO2-promoted urease activationat concentrations below that of Ni, whereas Mg and Ca ions did notinhibit this process. Ni-inhibitedspecies recovered their ability to be partially activated after EDTAtreatment. In contrast, samples thatwere exposed to Co or Cu ions were irreversibly inactivated, and EDTAtreatment of Zn- or Mn-inhibitedsamples led to reduced levels of activation competence.Mn-substituted urease, generated from ureaseapoprotein samples in a Mn- and CO2-dependent manner, wasshown to be active, whereas other metal-substituted forms of urease lacked activity. The Mn-proteinpossessed only 2% of the activity of Ni-activated apoprotein [∼8.0 vs ∼400 μmol min-1 (mgof protein)-1], but its KM valuewas only moderatelyaltered from that of the native enzyme (3.86 ± 0.15 mM vs 2.3 ± 0.2mM). Unlike the Ni-containingenzyme, Mn-urease was inhibited by EDTA. Given the evidence thaturease apoprotein binds numerousmetal ions, we speculate on possible roles for the UreD, UreF, and UreGaccessory proteins in ureaseactivation.
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