A protein catalytic framework with an N-terminal nucleophile is capable of self-activation

Brannigan, J.A.; Dodson, Guy; Duggleby, Helen J.; Moody, P.C.E.; Smith, Janet L.; Tomchick, Diana R.; Murzin, Alexey G.

doi:10.1038/378416a0

Cited by 599 publications

(496 citation statements)

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“…7). Therefore, it is likely that hGGT2 has acquired additional substitutions which contribute to its inability to fold properly or autocleave similar to hGGT1 and other members of the Ntn hydrolyase family (4). In addition to the sequence deviations documented earlier, hGGT2 propeptides also contain several conserved substitutions with unknown functional implications in the sequence spanning residues 421 to 435, using hGGT2-1 as the reference sequence (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

West

Wickham

Parks

et al. 2013

Antioxidants & Redox Signaling

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Aims: Human c-glutamyltranspeptidase 1 (hGGT1) is a cell-surface enzyme that is a regulator of redox adaptation and drug resistance due to its glutathionase activity. The human GGT2 gene encodes a protein that is 94% identical to the amino-acid sequence of hGGT1. Transcriptional profiling analyses in a series of recent publications have implicated the hGGT2 enzyme as a modulator of disease processes. However, hGGT2 has never been shown to encode a protein with enzymatic activity. The aim of this study was to express the protein encoded by hGGT2 and each of its known variants and to assess their stability, cellular localization, and enzymatic activity. Results: We discovered that the proteins encoded by hGGT2 and its variants are inactive propeptides. We show that hGGT2 cDNAs are transcribed with a similar efficiency to hGGT1, and the expressed propeptides are N-glycosylated. However, they do not autocleave into heterodimers, fail to localize to the plasma membrane, and do not metabolize c-glutamyl substrates. Substituting the coding sequence of hGGT1 to conform to alterations in a CX 3 C motif encoded by hGGT2 mRNAs disrupted autocleavage of the hGGT1 propeptide into a heterodimer, resulting in loss of plasma membrane localization and catalytic activity. Innovation and Conclusions: This is the first study to evaluate hGGT2 protein. The data show that hGGT2 does not encode a functional enzyme. Microarray data which have reported induction of hGGT2 mRNA should not be interpreted as induction of a protein that has a role in the metabolism of extracellular glutathione and in maintaining the redox status of the cell.

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Section: Discussionmentioning

confidence: 99%

“…Similar to hGGT1, hGGT2 is predicted to be a member of the N-terminal nucleophile (Ntn) family of hydrolases and, as a result, would require an autocatalytic cleavage for its functional activation (4,36). Characteristic of Ntn hydrolases, hGGT1 is initially expressed as a propeptide that is composed of 569 amino acids.…”

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confidence: 99%

Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

West

Wickham

Parks

et al. 2013

Antioxidants & Redox Signaling

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Section: ! Introductionmentioning

confidence: 99%

“…Only recently, two other threonine amidohydrolases, 20S proteasome [14] and aspartylglucosaminidase [15], have been reported. Both enzymes belong to the nonrelated superfamily of N-terminal nucleophile (Ntn) hydrolases [16].So far, neither kinetic experiments nor structural data could unambiguously identify which of the two threonine residues in asparaginase adjacent to the substrate is the primary nucleophile. Here we present the structure of E. coli asparaginase II T89V mutant with aspartate covalently bound to Thr-12, strongly suggesting that Thr-12 assumes the role of the primary nucleophile.…”

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confidence: 99%

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Palm¹,

Łubkowski²,

Derst³

et al. 1996

FEBS Letters

112

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Escherichia coli asparaginase |I catalyzes the hydrol-) sis of L-asparagine to L-aspartate via a threonine-bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.:£ey words'." Asparaginase II; Acyl-enzyme intermediate; !'hreonine amidohydrolase; Enzymatic mechanism !. IntroductionAsparaginases catalyze the hydrolysis of L-asparagine to L~spartate and ammonia. The enzymes isolated from Escheri-'hia coli (EcA) and Erwinia chrysanthemi (ERA) have been ltilized as anti-leukemia drugs for many years [1]. Treatment vith asparaginases is often accompanied by severe side effects, vhich are partially attributed to the glutaminase activity of hese enzymes [2]. In order to understand the enzyme specifi-:ity and to ultimately eliminate the glutaminase activity, the nechanism of action must be elucidated. Several members of t larger family of homologous L-asparaginases have thus been horoughly investigated over many years.Results of kinetic measurements [3,4] indicated that the en-,ymatic reaction proceeds via a covalent intermediate, probtbly a ~-aspartyl enzyme (Fig. 1). This mechanism was con-,irmed by NMR studies with aspartate through the oxygen ~xchange reaction with bulk solvent at the side chain carboxdate [5]. These experiments showed that at pH < 5 L-aspartate ~ith a protonated side chain binds to the enzyme as tightly as ~-asparagine and it can also form an acyl-enzyme intermediate, subsequently hydrolyzed to L-aspartate. Thus, at low pH 3oth L-aspartate and L-asparagine can function as substrates. l'he nature and identity of the primary nucleophile of the mzyme participating in the formation of the acyl-enzyme in-*Corresponding author. Fax: (1) . Each has the same tetrameric quaternary structure as it has in solution, a homotetramer of approx. 4x330 residues. The tetramer is more accurately described as a dimer of dimers. Each of two identical active sites in each dimer is formed by both monomers. In the structure the active sites have been observed with and without aspartate as ligand. Part of the active site is covered by a flexible loop that contains the two important residues Thr-12 and Tyr-25. The hydroxyl groups of Thr-12 and Thr-89 are closest to the side chain carboxylate of an aspartate bound in the active site. These residues are the most likely candidates for the primary nucleophile. Bacterial L-asparaginases were the first threonine amidohydrolases described in the literature. Only recently, two other threonine amidohydrolases, 20S proteasome [14] and aspartylglucosaminidase [15], have been reported. Both enzymes belong t...

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“…CA hydrolyzes Gl-7-ACA to 7-ACA (glutaryl 7-aminocephalosporanic acid to 7-aminocephalosporanic acid), which is a key intermediate for the synthesis of cephem antibiotics (4). Ntn hydrolases feature an ␣␤␤␣ sandwich and an N-terminal, catalytically active, nucleophile that is released by the first proteolytic step (5)(6)(7). Examples of Ntn hydrolases are glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (8), proteasome ␤-subunits (9), penicillin acylases (7), glucosamine-6-phosphate synthase (10), isoaspartyl aminopeptidase (11), taspase 1 (12), glycosylasparaginase (GA) (13,14), the human asparaginase-like protein 1 (hASRGL1) (15), Helicobacter pylori ␥-glutamyltranspeptidase (16), N-acylhomoserine lactone acylase PvdQ (PvdQ) (17), and cephalosporin acylases (18) whose crystal structures are available.…”

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confidence: 99%

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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A protein catalytic framework with an N-terminal nucleophile is capable of self-activation

Cited by 599 publications

References 28 publications

Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

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

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