PDBsum entry: 1hlw

Transferase

PDB id: 1hlw

Contents

Protein chain

150 a.a. *

Waters ×126

* Residue conservation analysis

PDB id:

1hlw

Name:

Transferase

Title:

Structure of the h122a mutant of the nucleoside diphosphate

Structure:

Nucleoside diphosphate kinase. Chain: a. Engineered: yes. Mutation: yes

Source:

Dictyostelium discoideum. Organism_taxid: 44689. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Hexamer (from PDB file)

Resolution:

1.90Å R-factor: 0.166 R-free: 0.208

Authors:

S.J.Admiraal,P.Meyer,B.Schneider,D.Deville-Bonne,J.Janin,D.H

Key ref:

S.J.Admiraal et al. (2001). Chemical rescue of phosphoryl transfer in a cavity mutant: a cautionary tale for site-directed mutagenesis. Biochemistry, 40, 403-413. PubMed id: 11148034 DOI: 10.1021/bi002472w

Date:

04-Dec-00 Release date: 28-Feb-01

Protein chain

P22887  (NDKC_DICDI) -  Nucleoside diphosphate kinase, cytosolic

Seq: 155 a.a.

Struc: 150 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.2.7.4.6 - Nucleoside-diphosphate kinase.
• Reaction: ATP + nucleoside diphosphate = ADP + nucleoside triphosphate

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

 Gene Ontology (GO) functional annotation 

• Cellular component: plasma membrane (6 terms)
• Biological process: cytoskeleton organization (13 terms)
• Biochemical function: nucleotide binding (6 terms)

reference

DOI no: 10.1021/bi002472w

Biochemistry 40:403-413 (2001)

PubMed id: 11148034

Chemical rescue of phosphoryl transfer in a cavity mutant: a cautionary tale for site-directed mutagenesis.

S.J.Admiraal, P.Meyer, B.Schneider, D.Deville-Bonne, J.Janin, D.Herschlag.

ABSTRACT

We have explored the ability of a nucleoside diphosphate kinase (NDPK) mutant in which the nucleophilic histidine has been replaced by glycine (H122G) to transfer phosphate from ATP to alcohols of varying pK(a), size, shape, and polarity. This cavity mutant does indeed act as a primitive alcohol kinase. The rate of its phosphoryl transfer to alcohols varies considerably, with values spanning a DeltaDeltaG(double dagger) range of 4 kcal/mol, whereas the alcohols have very similar intrinsic reactivities. Analysis of these results suggests that the ability to carry out phosphoryl transfer within the cavity is not a simple function of being small enough to enter the cavity, but rather is a complex function of steric, solvation, entropic, van der Waals packing, and electrostatic properties of the alcohol. In addition, large differences are observed between the reactivities of alcohols within the nucleophile cavity of H122G and the reactivities of the same alcohols within the nucleophile cavity of H122A, a mutant NDPK that differs from H122G by a single methyl group within the cavity. The crystal structures of the two cavity mutants are very similar to one another and to wild-type NDPK, providing no evidence for a structurally perturbed active site. The differences in reactivity between the two mutant proteins illustrate a fundamental limitation of energetic analysis from site-directed mutagenesis: although removal of a side chain is generally considered to be a conservative change, the energetic effects of any given mutation are inextricably linked to the molecular properties of the created cavity and the surrounding protein environment.