Uncoupling intramolecular processing and substrate hydrolysis in the N-terminal nucleophile hydrolase hASRGL1 by circular permutation.
The human asparaginase-like protein 1 (hASRGL1) catalyzes the hydrolysis of l-asparagine and isoaspartyl-dipeptides. As an N-terminal nucleophile (Ntn) hydrolase superfamily member, the active form of hASRGL1 is generated by an intramolecular cleavage step with Thr168 as the catalytic residue. However, in vitro, autoprocessing is incomplete (~50%), fettering the biophysical characterization of hASRGL1. We circumvented this obstacle by constructing a circularly permuted hASRGL1 that uncoupled the autoprocessing reaction, allowing us to kinetically and structurally characterize this enzyme and the precursor-like hASRGL1-Thr168Ala variant. Crystallographic and biochemical evidence suggest an activation mechanism where a torsional restraint on the Thr168 side chain helps drive the intramolecular processing reaction. Cleavage and formation of the active site releases the torsional restriction on Thr168, which is facilitated by a small conserved Gly-rich loop near the active site that allows the conformational changes necessary for activation.
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, United States.
Wenzong
W
Li
Jason R
JR
Cantor
S D
SD
Yogesha
Shirley
S
Yang
Lynne
L
Chantranupong
June Qingxia
JQ
Liu
Giulia
G
Agnello
George
G
Georgiou
Everett M
EM
Stone
Yan
Y
Zhang
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation
D001324:Autoantigens; D001216:Asparagine; D000581:Amidohydrolases; C466840:ASRGL1 protein, human; C438834:N-terminal nucleophile hydrolase; D001215:Asparaginase