Post‐translational processing and Thr‐206 are required for glycosylasparaginase activity

Fisher, Krishna J.; Klein, Melissa; Park, Hye Jeong; Vettese, Michelle B.; Aronson, Nathan N.

doi:10.1016/0014-5793(93)81355-4

Cited by 20 publications

(17 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thr-152 is also the key catalytic amino acid in autoproteolysis because it nucleophilically attacks the adjacent Asp-151, allowing the peptide bond between them to be broken and an active enzyme to be formed with a free ␣-amino group at the N terminus of the ␤ subunit (12,17). Previous mutagenesis studies on the human enzyme indicated that reductions of glycosylasparaginase activity in many cases are due to a slowing down of precursor autoproteolysis such that the enzyme is incompletely activated (13,14,24). This complexity in glycosylasparaginase processing creates experimental difficulties when investigating the function of specific amino acids in the mechanism of enzyme catalysis.…”

Section: Discussionmentioning

confidence: 99%

“…Based on its crystal structure, amino acid mutagenesis studies (13,16,17), and molecular dynamics calculations (30), glycosylasparaginase has a similar catalytic mechanism (Fig. 4).…”

Section: Discussionmentioning

confidence: 99%

“…After translation, the nascent single polypeptide is cleaved into two nonidentical ␣ and ␤ subunits by an autoproteolytic procedure (12). This self-cleavage produces the catalytically active glycosylasparaginase heterodimer (13,14). Glycosylasparaginases from all the different species show a high sequence homology and conservation of biochemical properties (9,15), and they are expected to share a similar threedimensional structure.…”

Section: Glycosylasparaginase (Glycoasparaginase Nmentioning

confidence: 99%

See 2 more Smart Citations

Site-directed Mutagenesis of Essential Residues Involved in the Mechanism of Bacterial Glycosylasparaginase

Liu

Guan

Aronson

1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Flavobacterium glycosylasparaginase was cloned in an Escherichia coli expression system. Site-directed mutagenesis was performed at residues suggested to be important in the catalytic mechanism based on the crystal structure of the human enzyme and other biochemical studies. In vitro autoproteolysis allowed the mutant enzymes to be activated, including those that were slow to self-cleave. Based on the activity of the mutant enzymes, six catalytically essential amino acids were identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183. Kinetic analysis of each mutant further defined the function of these residues in substrate specificity and reaction rate. Mutagenesis of the N-terminal nucleophile residue Thr-152 confirmed the key function of its side-chain hydroxyl group. Partial activities of mutants T152S/C were in agreement with the general mechanism of N-terminal nucleophile (Ntn)-amidohydrolases. The side-chain hydroxyl of Thr-170 contributes to the reaction rate based on studies of mutants T170S/C/A. Residues Asp-183 and Arg-180 were found to H-bond, respectively, with the charged ␣-amino and ␣-carboxyl group of the substrate (Asn-GlcNAc). Mutants R180Q/L and D183E/N had greatly decreased substrate affinity and reduced reaction rates. Kinetic studies also showed that Trp-11 is involved in regulation of the enzyme reaction rate, contradictory to a previous suggestion that this residue is involved in substrate binding. Asp-66 is a new residue found to be important in enzyme activity. The overall active site structure involving these catalytic residues resembles the glutaminase domain of glucosamine 6-phosphate synthase, another member of the Ntnamidohydrolase family of enzymes.

show abstract

Section: Discussionmentioning

confidence: 99%

“…Based on its crystal structure, amino acid mutagenesis studies (13,16,17), and molecular dynamics calculations (30), glycosylasparaginase has a similar catalytic mechanism (Fig. 4).…”

Section: Discussionmentioning

confidence: 99%

Section: Glycosylasparaginase (Glycoasparaginase Nmentioning

confidence: 99%

See 1 more Smart Citation

Site-directed Mutagenesis of Essential Residues Involved in the Mechanism of Bacterial Glycosylasparaginase

Liu

Guan

Aronson

1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Since glycosylasparaginase does not possess protease activity per se, the activation cannot be an autocatalytic process. It is a spontaneous intramolecular reaction with the key Based on the data presented here and in other published studies (5,6), we propose the following model of autoproteolysis of glycosylasparaginase, illustrated in Fig. 7.…”

Section: Discussionmentioning

confidence: 99%

Activation of Glycosylasparaginase

Guan

Cui

Rao

et al. 1996

Journal of Biological Chemistry

144

View full text Add to dashboard Cite

The activation mechanism of glycosylasparaginase of Flavobacterium meningosepticum has been analyzed by site-directed mutagenesis and activation of purified precursors in vitro. Mutation of Thr-152 to Ser or Cys leads to gene products that are not activated in vivo but are activated in vitro because processing of the mutant precursors is inhibited by certain amino acids in the cell. Kinetic studies reveal that activation is an intramolecular autoproteolytic process. The involvement of His-150 and Thr/Ser/Cys-152 in activation suggests that autoproteolysis resembles proteolysis by serine/cysteine proteases. Multiple functions of the highly conserved active threonine residue are implicated.

show abstract

“…Cleavage is essential for GA hydrolase activity and occurs at the amide bond in the consensus site between residues Asp151 and Thr152 (Ikonen et al, 1993;Fisher et al, 1993;Tarentino et al, 1995). This conserved threonine is thought to play central roles in both hydrolase activity (Kaartinen et al, 1991;Fisher et al, 1993) and autoproteolysis (Guan et al, 1996). Like other recently discovered protein autoprocessing pathways, GA self-catalyzes peptide-bond rearrangement through an N3O or N3S acyl shift (Perler, 1998a;Paulus, 1998).…”

Section: Introductionmentioning

confidence: 99%

Purification and crystallization of precursors and autoprocessed enzymes of Flavobacterium glycosylasparaginase: an N-terminal nucleophile hydrolase

Cui¹,

Liao²,

Guan³

et al. 1999

Acta Crystallogr D Biol Cryst

View full text Add to dashboard Cite

Glycosylasparaginase (GA) represents a novel group of proteins that are activated by self-catalyzed peptide-bond cleavage from a single-chain precursor to yield the two subunits required for hydrolase activity. The wild-type GA precursor autoproteolyzes spontaneously into α and β subunits. Strategies are reported here for purification to homogeneity of GA from Flavobacterium meningosepticum in both single-chain precursor and mature (autoprocessed) forms. The recombinant proteins crystallize in different space groups: P1 and P21 for the precursor and mature enzymes, respectively. The precursor crystals diffract to 1.9 Å resolution with laboratory X-ray radiation.

show abstract

Post‐translational processing and Thr‐206 are required for glycosylasparaginase activity

Cited by 20 publications

References 26 publications

Site-directed Mutagenesis of Essential Residues Involved in the Mechanism of Bacterial Glycosylasparaginase

Site-directed Mutagenesis of Essential Residues Involved in the Mechanism of Bacterial Glycosylasparaginase

Activation of Glycosylasparaginase

Purification and crystallization of precursors and autoprocessed enzymes of Flavobacterium glycosylasparaginase: an N-terminal nucleophile hydrolase

Contact Info

Product

Resources

About