The role of phosphagen specificity loops in arginine kinase

Arezki Azzi, Shawn A. Clark, W. Ross Ellington, Michael S. Chapman

Research output: Contribution to journalArticlepeer-review

58 Scopus citations


Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.

Original languageEnglish (US)
Pages (from-to)575-585
Number of pages11
JournalProtein Science
Issue number3
StatePublished - Mar 2004
Externally publishedYes


  • Creatine kinase
  • Kinetics
  • Mutation
  • Phosphagen kinase
  • Structure
  • Substrate specificity

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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