Temperature Dependence of the Enzyme-Substrate Recognition Mechanism

Ura, H.; Harata, Kazuaki; Matsui, Ichiro; Kuramitsu, Seiki

doi:10.1093/oxfordjournals.jbchem.a002829

Cited by 10 publications

(6 citation statements)

References 4 publications

(4 reference statements)

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“…The thermophilic enzymes show increased rigidity at room temperature compared with their mesophilic counterparts (28 -31). On the basis of x-ray structures of mesophilic and thermophilic enzymes (32)(33)(34)(35)(36), it was suggested that the thermostable enzymes show a smaller domain movement compared with mesophilic ones (32). This assumption seems to be applicable to the behavior of the ATP binding domain of tAsS, which is different from that of eAsS.…”

Section: Resultsmentioning

confidence: 99%

Structures of Argininosuccinate Synthetase in Enzyme-ATP Substrates and Enzyme-AMP Product Forms

Goto

Omi

Miyahara

et al. 2003

Journal of Biological Chemistry

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In prokaryotes, an arginine is synthesized by eight-step reactions (arginine biosynthetic pathway) using glutamate as a starting material (1). The glutamate is converted to ornithine by five-step reactions, and the ornithine then enters into the urea cycle to produce arginine in three-step reactions. Argininosuccinate synthetase (AsS) 1 catalyzes the seventh step of the arginine biosynthesis (the second step of the urea cycle). AsS reversibly catalyzes the adenosine triphosphate (ATP)-dependent condensation of citrulline with aspartate (Scheme 1).It has been proposed that GMP synthetase, NAD ϩ synthetase, asparagine synthetase, and AsS have a common domain belonging to a new family of "N-type" ATP pyrophosphatases (4, 5). The domain has the modified version of the P-loop (PPloop) of H-Ser-Gly-Gly-X-Asp-Ser/Thr-Ser/Thr (where H is any hydrophobic amino acid and X is any amino acid) specific for a pyrophosphate. X-ray structures of GMP synthetase (4), NAD ϩ synthetase (6 -8), and asparagine synthetase (9, 10) show that ATP binding domains are folded into the same open ␣/␤ structure, and pyrophosphate and the ␤-and ␥-phosphates of ATP interact with the PP-loop in GMP synthetase and NAD ϩ synthetase, respectively.For the first time Escherichia coli AsS (eAsS) and its complex with citrulline and aspartate have been determined as the x-ray structure of AsS (11). The enzyme consists of the ATP binding domain (small domain) and the synthetase domain (large domain). The ATP binding domain of eAsS has the same fold as that of a new family of N-type ATP pyrophosphatases. A corollary of the ATP binding model to the active site is that a conformational change in the enzyme is necessary for the catalytic reaction. To confirm this proposal, the structures of eAsS⅐ATP and eAsS⅐ATP⅐citrulline have been determined by x-ray methods (12). Comparisons of these two complexes with eAsS and eAsS⅐citrulline⅐aspartate revealed that ATP binding induces a rotation of the ATP binding domain toward the synthetase domain. Based on the structural elucidation of eAsS complexes, the observed kinetic properties were explained, and the catalytic mechanism of AsS was proposed.The structures of Thermus thermophilus HB8 AsS (tAsS), tAsS⅐ATP, and tAsS⅐AMP-PNP⅐arginine⅐succinate have been previously determined by us (13) to show an overall structure similar to that of eAsS. No conformational change in the ATP binding domain was observed on binding of ATP and AMP-PNP, implying that the reaction may proceed without the conformational change at the molecular level. ATP (or AMP-PNP) and substrate analogues are bound to the active site with their reaction sites close to one another and located in a geometric orientation favorable to the catalytic action. The mechanism of the reaction was proposed on the basis of the enzyme-substrate complex model.We have determined the structures of tAsS in the complex with intact ATP and substrates (citrulline and aspartate), in the complex with AMP and product (argininosuccinate), and in the complex with AMP-PNP, substrat...

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Section: Resultsmentioning

confidence: 99%

Structures of Argininosuccinate Synthetase in Enzyme-ATP Substrates and Enzyme-AMP Product Forms

Goto

Omi

Miyahara

et al. 2003

Journal of Biological Chemistry

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“…Internal studies have shown that SMART digest trypsin gradually becomes inactive at 75 °C and compared to conventional MAM digestion workflows, SMART digest workflows appeared to have higher levels of missed cleavages (data not shown). While some missed cleavages are commonly observed, it was hypothesized that elevated levels of missed cleavages in SMART digest trypsin most likely stemmed from the high temperature the digestion was performed at, as this may impact enzyme substrate specificity. , Furthermore, the gradual inactivation of SMART digest trypsin at high temperatures would also play a part. Based on these considerations, it was decided to evaluate a two-step SMART digest trypsin workflow in which the first step was performed at the usual high temperature, but for a shorter period, and the second step was performed at lower temperature using fresh SMART digest trypsin resin.…”

Section: Resultsmentioning

confidence: 99%

“…While some missed cleavages are commonly observed, it was hypothesized that elevated levels of missed cleavages in SMART digest trypsin most likely stemmed from the high temperature the digestion was performed at, as this may impact enzyme substrate specificity. 20,21 Furthermore, the gradual inactivation of SMART digest trypsin at high temperatures would also play a part. Based on these considerations, it was decided to evaluate a two-step *Each identified charge state contributes to the peptide count.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides

et al. 2022

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Peptide mapping by liquid chromatography mass spectrometry (LC-MS) and the related multi-attribute method (MAM) are well-established analytical tools for verification of the primary structure and mapping/quantitation of co-and posttranslational modifications (PTMs) or product quality attributes in biopharmaceutical development. Proteolytic digestion is a key step in peptide mapping workflows, which traditionally is laborintensive, involving multiple manual steps. Recently, simple hightemperature workflows with automatic digestion were introduced, which facilitate robustness and reproducibility across laboratories. Here, a modified workflow with an automatic digestion step is presented, which includes a two-step digestion at high and low temperatures, as opposed to the original one-step digestion at a high temperature. The new automatic digestion workflow significantly reduces the number of missed cleavages, obtaining a more complete digestion profile. In addition, we describe how chromatographic peak tailing and carry-over is dramatically reduced for hydrophobic peptides by switching from the traditional C18 reversed-phase (RP) column chemistry used for peptide mapping to a less retentive C4 column chemistry. No negative impact is observed on MS/MS-derived sequence coverage when switching to a C4 column chemistry. Overall, the new peptide mapping workflow significantly reduces the number of missed cleavages, yielding more robust and simple data interpretation, while providing dramatically reduced tailing and carry-over of hydrophobic peptides.

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“…The five closest structural homologues of LiTAT were identified using the DaliLite v.3 server (Holm & Rosenströ m, 2010): TcTAT (PDB entry 1bw0; Z-score 59.2, 44% identity; Blankenfeldt et al, 1999), TAT from Homo sapiens (PDB entry 3dyd; Z-score 54.4, 37% identity; Structural Genomics Consortium, unpublished work), alanine aminotransferase from Pyrococcus furiosus (PDB entry 1xi9; Z-score 53.1, 28% identity; Southeast Collaboratory for Structural Genomics, unpublished work), TAT from Mus musculus (PDB entry 3pdx; Z-score 52.8, 38% identity; Mehere et al, 2010) and -aminotransferase from P. horikoshii (PDB entry 1gd9; Z-score 48.4, 22% identity; Ura et al, 2001). These alignments show the low percentage similarity between LiTAT and the other closely related orthologues.…”

Section: Crystal Structure Of Litatmentioning

confidence: 99%