1EJU

CRYSTAL STRUCTURE OF THE H320N VARIANT OF KLEBSIELLA AEROGENES UREASE

Summary for 1EJU

• Entry DOI: 10.2210/pdb1eju/pdb
• Related: 1EJR 1EJS 1EJT 1EJV 1EJW 1EJX
• Descriptor: UREASE ALPHA SUBUNIT, UREASE BETA SUBUNIT, UREASE GAMMA SUBUNIT, ... (5 entities in total)
• Functional Keywords: alpha-beta barrel, nickel metalloenzyme, hydrolase
• Biological source: Klebsiella aerogenes; More
• Cellular location: Cytoplasm (By similarity): P18314 P18315 P18316
• Total number of polymer chains: 3
• Total formula weight: 82729.32

Authors

Pearson, M.A.,Park, I.S.,Schaller, R.A.,Michel, L.O.,Karplus, P.A.,Hausinger, R.P. (deposition date: 2000-03-04, release date: 2000-09-08, Last modification date: 2021-11-03)

Primary citation

Pearson, M.A.,Park, I.S.,Schaller, R.A.,Michel, L.O.,Karplus, P.A.,Hausinger, R.P.
Kinetic and structural characterization of urease active site variants.
Biochemistry, 39:8575-8584, 2000

PubMed Abstract: Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-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 approximately 10(-)(5)-fold deficiencies in rates, modest K(m) 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 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-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-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.

PubMed: 10913264
DOI: 10.1021/bi000613o

Experimental method

X-RAY DIFFRACTION (2 Å)