Characterization and functional analysis of the cis-autoproteolysis active center of glycosylasparaginase.
Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing. This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids. The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation. Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180. The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.
Guan
C
C
New England Biolabs Inc., Beverly, Massachusetts 01915, USA.
Liu
Y
Y
Shao
Y
Y
Cui
T
T
Liao
W
W
Ewel
A
A
Whitaker
R
R
Paulus
H
H
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001120:Arginine; D001224:Aspartic Acid; D001227:Aspartylglucosylaminase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008956:Models, Chemical; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D001224:Aspartic Acid; D001120:Arginine; D001227:Aspartylglucosylaminase