The active site cysteine of arginine kinase

Structural and functional analysis of partially active mutants

James L. Gattis, Eliza Ruben, Marcia O. Fenley, W. Ross Ellington, Michael Chapman

Research output: Contribution to journalArticle

33 Citations (Scopus)

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 Å 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 Å of the nonreactive 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 to enhance the catalytic rate through electrostatic stabilization of the transition state.

Original languageEnglish (US)
Pages (from-to)8680-8689
Number of pages10
JournalBiochemistry
Volume43
Issue number27
DOIs
StatePublished - Jul 13 2004
Externally publishedYes

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Arginine Kinase
Functional analysis
Structural analysis
Cysteine
Catalytic Domain
Substrates
Asparagine
Catalysis
Aspartic Acid
Alanine
Adenosine Diphosphate
Serine
Arginine
Chlorides
Adenosine Triphosphate
Horseshoe Crabs
Enzymes
Static Electricity
Sulfur
Nitrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

The active site cysteine of arginine kinase : Structural and functional analysis of partially active mutants. / Gattis, James L.; Ruben, Eliza; Fenley, Marcia O.; Ellington, W. Ross; Chapman, Michael.

In: Biochemistry, Vol. 43, No. 27, 13.07.2004, p. 8680-8689.

Research output: Contribution to journalArticle

Gattis, James L. ; Ruben, Eliza ; Fenley, Marcia O. ; Ellington, W. Ross ; Chapman, Michael. / The active site cysteine of arginine kinase : Structural and functional analysis of partially active mutants. In: Biochemistry. 2004 ; Vol. 43, No. 27. pp. 8680-8689.
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