Expression, Purification, Assay, and Crystal Structure of Perdeuterated Human Arginase I

Arch Biochem Biophys. 2007 Sep 1;465(1):82-9. doi: 10.1016/j.abb.2007.04.036. Epub 2007 May 21.

Abstract

Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to yield l-ornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2(S)-amino-6-boronohexanoic acid (ABH) at 1.90A resolution. Prior to the neutron diffraction experiment, it is important to establish that perdeuteration does not cause any unanticipated structural or functional changes. Accordingly, we find that perdeuterated human arginase I exhibits catalytic activity essentially identical to that of the unlabeled enzyme. Additionally, the structure of the perdeuterated human arginase I-ABH complex is identical to that of the corresponding complex with the unlabeled enzyme. Therefore, we conclude that crystals of the perdeuterated human arginase I-ABH complex are suitable for neutron crystallographic study.

Publication types

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

MeSH terms

  • Arginase / chemistry*
  • Arginase / ultrastructure*
  • Computer Simulation
  • Deuterium / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Models, Chemical*
  • Models, Molecular*
  • Protein Conformation
  • Protein Engineering
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Substrate Specificity

Substances

  • Recombinant Proteins
  • Deuterium
  • Arginase