Activation of Glycosylasparaginase

Guan, Chaowen; Cui, Tao; Rao, Vibha; Liao, Wei; Benner, Jack S.; Lin, Ching-Lun; Comb, Donald G.

doi:10.1074/jbc.271.3.1732

Cited by 144 publications

(104 citation statements)

References 23 publications

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“…The hydroxyl group of Thr152 is believed to be the nucleophile in both autoproteolysis and hydrolase catalysis (Guan et al, 1996), suggesting an overlap between these two activity centres. We therefore made several point mutations at the functional groups proposed to be involved in the hydrolase activity (Oinonen et al, 1995;Guan et al, 1996;Guo et al, 1998). For example, point mutants were generated at Thr152 (named T152X, where X stands for the substituting residue) and at Trp11 (named W11F, where Trp11 has been substituted with a phenylalanine).…”

Section: Resultsmentioning

confidence: 99%

“…Point mutations at the active site were generated as described previously (Guan et al, 1996;Liu et al, 1998). With the aid of the structure of the mature enzyme (Guo et al, 1998), residues surrounding Thr152 were chosen for site-directed mutagenesis.…”

Section: Expression and Puri®cationmentioning

confidence: 99%

“…Another intriguing aspect of this enzyme is that a single-chain precursor is processed by intramolecular proteolysis to generate the newly exposed N-terminal threonine and an active hydrolase with and subunits (Guan et al, 1996). 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).…”

Section: Introductionmentioning

confidence: 99%

“…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%

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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

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Expression and Puri®cationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

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“…Autoproteolysis in the latter mutant was also very slow but it could be accelerated by hydroxylamine (44). This inactivation was possibly due to the slight deviation of the nucleophile group in the crystal structure (20).…”

Section: Substitution Of the Ntn Serine Simultaneously Affected The Smentioning

confidence: 95%

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Yin

Deng

Zhao

et al. 2011

Journal of Biological Chemistry

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Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature ␣␤ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu 159 and Gly 160 , and detected the corresponding 10-aa spacer 160 GDPPDLADQG 169 of CA mutants. The site of the second cleavage depended on Glu 159 : moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a firstorder, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser 170 of the ␤-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intramolecular process performed by an N-terminal nucleophile, during which large conformational changes in the ␣-subunit C-terminal region are required to bridge the gap between Glu 159 and Ser 170 .

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Protein-Spleißen: Mechanismus und Anwendungen

Noren

Wang

Perler³

2000

Angewandte Chemie

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Protein‐Spleißen, das Äquivalent des RNA‐Spleißens auf Proteinebene, ist ein neu entdeckter posttranslationaler Prozess, der über ein verzweigtes Protein‐Intermediat zur Bildung zweier verschiedener Polypeptide führt, die sich vom selben Gen ableiten. Die experimentellen Befunde zur Aufklärung des vollständigen Mechanismus des Protein‐Spleißens sowie zahlreiche Anwendungen für das Protein‐Engineering unter Verwendung von modifizierten Inteinen werden beschrieben, darunter die Einführung von α‐Thioesterbindungen in Proteine, wodurch die Beschränkungen der Festphasensynthese von Peptiden umgangen werden.

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Activation of Glycosylasparaginase

Cited by 144 publications

References 23 publications

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

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

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Protein-Spleißen: Mechanismus und Anwendungen

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