PDBsum entry 1ejv

Hydrolase

PDB id: 1ejv

Contents

Protein chains

553 a.a. *

101 a.a. *

100 a.a. *

Metals

_NI ×2

Waters ×406

* Residue conservation analysis

PDB id:

1ejv

Name:

Hydrolase

Title:

Crystal structure of the h320q variant of klebsiella aerogenes urease

Structure:

Urease alpha subunit. Chain: c. Engineered: yes. Mutation: yes. Urease beta subunit. Chain: b. Engineered: yes. Urease gamma subunit. Chain: a.

Source:

Klebsiella aerogenes. Organism_taxid: 28451. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Biol. unit:

Nonamer (from PDB file)

Resolution:

2.40Å R-factor: 0.181

Authors:

M.A.Pearson,I.S.Park,R.A.Schaller,L.O.Michel,P.A.Karplus, R.P.Hausinger

Key ref:

M.A.Pearson et al. (2000). Kinetic and structural characterization of urease active site variants. Biochemistry, 39, 8575-8584. PubMed id: 10913264 DOI: 10.1021/bi000613o

Date:

04-Mar-00 Release date: 08-Sep-00

Protein chain

P18314  (URE1_KLEAE) -  Urease subunit alpha from Klebsiella aerogenes

Seq: 567 a.a.

Struc: 553 a.a.*

Protein chain

P18315  (URE2_KLEAE) -  Urease subunit beta from Klebsiella aerogenes

Seq: 106 a.a.

Struc: 101 a.a.

Protein chain

P18316  (URE3_KLEAE) -  Urease subunit gamma from Klebsiella aerogenes

Seq: 100 a.a.

Struc: 100 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chains C, B, A: E.C.3.5.1.5 - urease.
• Reaction: urea + 2 H2O + H⁺ = hydrogencarbonate + 2 NH4⁺
• Cofactor: Ni(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1021/bi000613o

Biochemistry 39:8575-8584 (2000)

PubMed id: 10913264

Kinetic and structural characterization of urease active site variants.

M.A.Pearson, I.S.Park, R.A.Schaller, L.O.Michel, P.A.Karplus, R.P.Hausinger.

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.