1SD0

Structure of arginine kinase C271A mutant

Summary for 1SD0

• Entry DOI: 10.2210/pdb1sd0/pdb
• Related: 1BG0 1M15 1M80
• Descriptor: Arginine kinase, NITRATE ION, MAGNESIUM ION, ... (7 entities in total)
• Functional Keywords: essential, cysteine, bimolecular, phosphotransferase, transition state, chloride, phosphagen kinase, transferase
• Biological source: Limulus polyphemus (Atlantic horseshoe crab)
• Cellular location: Cytoplasm: P51541
• Total number of polymer chains: 1
• Total formula weight: 40969.88

Authors

Gattis, J.L.,Ruben, E.,Fenley, M.O.,Ellington, W.R.,Chapman, M.S. (deposition date: 2004-02-12, release date: 2004-07-27, Last modification date: 2023-08-23)

Primary citation

Gattis, J.L.,Ruben, E.,Fenley, M.O.,Ellington, W.R.,Chapman, M.S.
The active site cysteine of arginine kinase: structural and functional analysis of partially active mutants
Biochemistry, 43:8680-8689, 2004

PubMed 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.

PubMed: 15236576
DOI: 10.1021/bi049793i

Experimental method

X-RAY DIFFRACTION (2.3 Å)