Kinetic and structural characterization of urease active site variants.

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >1014-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site:  His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar ∼10-5-fold deficiencies in rates, modest Km changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 103-fold increased Km over that of native enzyme, whereas the increase is less marked (∼102-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-typ...