Post-translational processing and Thr-206 are required for glycosylasparaginase activity.
Lysosomal glycosylasparaginase is encoded as a 36.5 kDa polypeptide that is post-translationally processed to subunits of 19.5 kDa (heavy) and 15 kDa (light). Recombinant glycosylasparaginase has been expressed in Spodoptera frugiperda insect cells enabling the precursor and processed forms to be isolated and their catalytic potential determined. Only the subunit conformation was functional indicating glycosylasparaginase is encoded as an inactive zymogen. The newly created amino terminal residue of the light subunit following maturation, Thr-206, is believed to be involved in the catalytic mechanism [1992, J. Biol. Chem. 267, 6855-6858]. Here we have constructed two amino acid substitution mutants replacing Thr-206 with Ala-206 or Ser-206 and demonstrate that both destroy enzyme activity.
Fisher
K J
KJ
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Klein
M
M
Park
H
H
Vettese
M B
MB
Aronson
N N
NN
D000595:Amino Acid Sequence; D000818:Animals; D001227:Aspartylglucosylaminase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D003062:Codon / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013912:Threonine; D014162:Transfection
D003062:Codon; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013912:Threonine; D001227:Aspartylglucosylaminase