The differential ability of asparagine and glutamine in promoting the closed/active enzyme conformation rationalizes the Wolinella succinogenes L-asparaginase substrate specificity

Sci Rep. 2017 Jan 31;7:41643. doi: 10.1038/srep41643.

Abstract

Many side effects of current FDA-approved L-asparaginases have been related to their secondary L-glutaminase activity. The Wolinella succinogenes L-asparaginase (WoA) has been reported to be L-glutaminase free, suggesting it would have fewer side effects. Unexpectedly, the WoA variant with a proline at position 121 (WoA-P121) was found to have L-glutaminase activity in contrast to Uniprot entry P50286 (WoA-S121) that has a serine residue at this position. Towards understanding how this residue impacts the L-glutaminase property, kinetic analysis was coupled with crystal structure determination of these WoA variants. WoA-S121 was confirmed to have much lower L-glutaminase activity than WoA-P121, yet both showed comparable L-asparaginase activity. Structures of the WoA variants in complex with L-aspartic acid versus L-glutamic acid provide insights into their differential substrate selectivity. Structural analysis suggests a mechanism by which residue 121 impacts the conformation of the conserved tyrosine 27, a component of the catalytically-important flexible N-terminal loop. Surprisingly, we could fully model this loop in either its open or closed conformations, revealing the roles of specific residues of an evolutionary conserved motif among this L-asparaginase family. Together, this work showcases critical residues that influence the ability of the flexible N-terminal loop for adopting its active conformation, thereby effecting substrate specificity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Amino Acids / metabolism
  • Asparaginase / chemistry*
  • Asparaginase / genetics
  • Asparaginase / metabolism*
  • Asparagine / chemistry
  • Asparagine / metabolism*
  • Conserved Sequence
  • Enzyme Activation
  • Glutaminase / chemistry*
  • Glutaminase / genetics
  • Glutaminase / metabolism*
  • Glutamine / chemistry
  • Glutamine / metabolism*
  • Kinetics
  • Models, Molecular
  • Point Mutation
  • Protein Conformation
  • Substrate Specificity
  • Wolinella / metabolism*

Substances

  • Amino Acids
  • Glutamine
  • Asparagine
  • Asparaginase
  • Glutaminase