| Abstract
| - Cys319 is located on a mobile flap covering the activesite of Klebsiella aerogenes urease butdoes not play an essential role in catalysis. Four urease variantsaltered at position C319 range fromhaving high activity (C319A) to no measurable activity (C319Y),indicating Cys is not required at thisposition, but its presence is highly influential [Martin, P. R., &Hausinger, R. P. (1992) J. Biol.Chem.267, 20024−20027]. Here, we present 2.0 Åresolution crystal structures of C319A, C319S, C319D, andC319Y proteins and the C319A variant inhibited by acetohydroxamic acid.These structures show changesin the hydration of the active site nickel ions and in the position andflexibility of the active site flap.The C319Y protein exhibits an alternate conformation of the flap,explaining its lack of activity. Thechanges in hydration and conformation suggest that there are suboptimalprotein−solvent and protein−protein interactions in the empty urease active site which contribute tourease catalysis. Specifically, wehypothesize that the suboptimal interactions may provide a significantsource of substrate binding energy,and such hidden energy may be a common phenomenon for enzymes thatcontain mobile active siteloops and undergo an induced fit. The acetohydroxamic acid-boundstructure reveals a chelate interactionsimilar to those seen in other metalloenzymes and in a small moleculenickel complex. The inhibitorbinding mode supports the proposed mode of urea binding. Wecomplement these structural studies withextended functional studies of C319A urease to show that it hasenhanced stability and resistance toinhibition by buffers containing nickel ions. The near wild-typeactivity and enhanced stability of theC319A variant make it useful for further studies of ureasestructure−function relationships.
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