1ejr: Difference between revisions

==Overview==

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.

==About this Structure==

1EJR is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Klebsiella_aerogenes Klebsiella aerogenes] with NI as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Urease Urease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.5.1.5 3.5.1.5] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1EJR OCA].

 

Kinetic and structural characterization of urease active site variants., Pearson MA, Park IS, Schaller RA, Michel LO, Karplus PA, Hausinger RP, Biochemistry. 2000 Jul 25;39(29):8575-84. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10913264 10913264]

[[Category: Protein complex]]

[[Category: Urease]]

[[Category: Hausinger, R.P.]]

[[Category: Karplus, P.A.]]

[[Category: Michel, L.O.]]

[[Category: Park, I.S.]]

[[Category: Pearson, M.A.]]

[[Category: Schaller, R.A.]]

[[Category: NI]]