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 2.3Å resolution, determined as a transition state analog 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 3.5Å of the non-reactive Nη 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 enhance the catalytic rate through electrostatic stabilization of the transition state.
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