PDBsum entry 1ejs

Hydrolase

PDB id: 1ejs

Contents

Protein chains

566 a.a. *

101 a.a. *

100 a.a. *

Metals

_NI ×2

Waters ×406

* Residue conservation analysis

References listed in PDB file

Key reference

• Title: Kinetic and structural characterization of urease active site variants.
• Authors: M.A.Pearson, I.S.Park, R.A.Schaller, L.O.Michel, P.A.Karplus, R.P.Hausinger.
• Ref.: Biochemistry, 2000, 39, 8575-8584. [DOI no: 10.1021/bi000613o]
• PubMed id: 10913264

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.