1SD0: Structure Of Arginine Kinase C271a Mutant

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.
PDB ID: 1SD0Download
MMDB ID: 28563
PDB Deposition Date: 2004/2/12
Updated in MMDB: 2007/10
Experimental Method:
x-ray diffraction
Resolution: 2.3  Å
Source Organism:
Similar Structures:
Biological Unit for 1SD0: monomeric; determined by author and by software (PISA)
Molecular Components in 1SD0
Label Count Molecule
Protein (1 molecule)
Arginine Kinase
Molecule annotation
Chemicals (5 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB