PDBsum entry 1sd0

Transferase

PDB id: 1sd0

Contents

Protein chain

356 a.a. *

Ligands

NO3

ARG

ADP

Metals

_MG

_CL

Waters ×288

* Residue conservation analysis

PDB id:

1sd0

Name:

Transferase

Title:

Structure of arginine kinase c271a mutant

Structure:

Arginine kinase. Chain: a. Synonym: ak. Engineered: yes. Mutation: yes

Source:

Limulus polyphemus. Atlantic horseshoe crab. Organism_taxid: 6850. Gene: ak. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.30Å R-factor: 0.209 R-free: 0.236

Authors:

J.L.Gattis,E.Ruben,M.O.Fenley,W.R.Ellington,M.S.Chapman

Key ref:

J.L.Gattis et al. (2004). The active site cysteine of arginine kinase: structural and functional analysis of partially active mutants. Biochemistry, 43, 8680-8689. PubMed id: 15236576 DOI: 10.1021/bi049793i

Date:

12-Feb-04 Release date: 27-Jul-04

Protein chain

P51541  (KARG_LIMPO) -  Arginine kinase from Limulus polyphemus

Seq: 357 a.a.

Struc: 356 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.3.3 - arginine kinase.
• Reaction: L-arginine + ATP = N(omega)-phospho-L-arginine + ADP + H⁺

L-arginine + ATP (Bound ligand (Het Group name = ARG) corresponds exactly) = N(omega)-phospho-L-arginine (Bound ligand (Het Group name = ADP) corresponds exactly) + ADP + H(+)

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

reference

DOI no: 10.1021/bi049793i

Biochemistry 43:8680-8689 (2004)

PubMed id: 15236576

The active site cysteine of arginine kinase: structural and functional analysis of partially active mutants.

J.L.Gattis, E.Ruben, M.O.Fenley, W.R.Ellington, M.S.Chapman.

ABSTRACT

Arginine kinase buffers cellular ATP levels by catalyzing reversible phosphoryl transfer between ATP and arginine. A conserved cysteine has long been thought important in catalysis. Here, cysteine 271 of horseshoe crab arginine kinase has been mutated to serine, alanine, asparagine, or aspartate. Catalytic turnover rates were 0.02-1.0% of wild type, but the activity of uncharged mutations could be partially rescued with chloride. Steady-state binding constants were slightly increased, more so for phospho-L-arginine than ADP. Substrate binding synergy observed in many phosphagen kinases was reduced or eliminated in mutant enzymes. The crystallographic structure of the alanine mutant at 2.3 A resolution, determined as a transition state analogue complex with arginine, nitrate, and MgADP, was nearly identical to wild type. Enzyme-substrate interactions are maintained as in wild type, and substrates remain at least roughly aligned for in-line phosphoryl transfer. Homology models with serine, asparagine, or aspartate replacing the active site cysteine similarly show only minor structural changes. Most striking, however, is the presence in the C271A mutant crystallographic structure of a chloride ion within 3.5 A of the nonreactive N(eta) substrate nitrogen, approximating the position of the sulfur in the wild-type's cysteine. Together, the results contradict prevailing speculation that the cysteine mediates a substrate-induced conformational change, confirm that it is the thiolate form that is relevant to catalysis, and suggest that one of its roles is to help to enhance the catalytic rate through electrostatic stabilization of the transition state.