Expression, purification, assay, and crystal structure of perdeuterated human arginase I.
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.
Di Costanzo
Luigi
L
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
Moulin
Martine
M
Haertlein
Michael
M
Meilleur
Flora
F
Christianson
David W
DW
D001119:Arginase / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
D011994:Recombinant Proteins; D003903:Deuterium; D001119:Arginase