Soybean trypsin inhibitor (Kunitz) and its complex with trypsin. Carbon-13 nuclear magnetic resonance studies of the reactive site arginine

Mw, Baillargeon; Laskowski, M.; De, Neves; Ma, Porubcan; Re, Santini; Jl, Markely

doi:10.1021/bi00566a006

Cited by 54 publications

(31 citation statements)

References 61 publications

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“…Early X-ray crystallographic studies [5,6] led to the claim that soya bean and bovine pancreatic trypsin inhibitors formed a tetrahedral adduct with trypsin [3]. However, 13 C-NMR studies did not substantiate this claim [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

13C- and 1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors

Malthouse

2007

Biochemical Society Transactions

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Catalysis by the serine proteinases proceeds via a tetrahedral intermediate whose oxyanion is stabilized by hydrogen-bonding in the oxyanion hole. There have been extensive (13)C-NMR studies of oxyanion and tetrahedral intermediate stabilization in trypsin, subtilisin and chymotrypsin using substrate-derived chloromethane inhibitors. One of the limitations of these inhibitors is that they irreversibly alkylate the active-site histidine residue which results in the oxyanion not being in the optimal position in the oxyanion hole. Substrate-derived glyoxal inhibitors are reversible inhibitors which, if they form tetrahedral adducts in the same way as substrates form tetrahedral intermediates, will overcome this limitation. Therefore we have synthesized (13)C-enriched substrate-derived glyoxal inhibitors which have allowed us to use (13)C-NMR and (1)H-NMR to determine how they interact with proteinases. It is hoped that these studies will help in the design of specific and highly potent warheads for serine proteinase inhibitors.

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Section: Introductionmentioning

confidence: 99%

13C- and 1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors

Malthouse

2007

Biochemical Society Transactions

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“…the cDNA clones encoding the precursors for Ti a and Ti b was reported (Song et al, 1991). The mechanism of inhibition of PPT by STI was proposed Baillargeon et al, 1980). A stable complex between the virgin inhibitor and trypsin is rapidly formed and dissociates very slowly into the free enzyme and a mixture of unmodi®ed and modi®ed inhibitors.…”

Section: Introductionmentioning

confidence: 99%

“…In the re®ned structures of complex between bovine pancreatic trypsin inhibitor (BPTI) and bovine pancreatic trypsin (BPT), the scissile peptide bond remains nearly intact, with a slight out-of-plane deformation of the carbonyl oxygen atom being observed (Bode & Huber, 1992). However, the absence of a large up®eld shift in the 13 C of the P1 residue of STI upon complex formation suggested that the STI:PPT complex is not a covalent, fully tetrahedral adduct (Baillargeon et al, 1980). More recently, no out-of-plane distortion around the inhibitor's scissile peptide bond was observed in the re®ned structure of the complex between porcine b-trypsin and MCTI-A, a trypsin inhibitor from the squash family (Huang et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator

Song

Suh

1998

Journal of Molecular Biology

229

162

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“…Two of the important results from these structural studies are that the reactive-site bonds of these inhibitors are not cleaved in an enzyme-inhibitor complex, and that there is a close approach of the 0" of the active site serine to the carbonyl-carbon atom of the P1 ¶ residue of the inhibitor (=2.7 A). A tetrahedral enzyme-inhibitor adduct is not formed (1,14,15).…”

mentioning

confidence: 98%

Crystal and molecular structure of chymotrypsin inhibitor 2 from barley seeds in complex with subtilisin Novo

McPhalen

Svendsen

Jonassen³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

117

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The serine proteinase inhibitor from barley seeds, chymotrypsin inhibitor 2 (CI-2), has been crystallized in a molecular complex with subtilisin Novo (EC 3.4.21.14). The crystal structure of this complex has been determined at 2.1-A resolution by the molecular replacement method and partially refined by restrained-parameter least-squares methods. The present crystallographic R factor (ZIIFoI -IFcII/ZIFoI) is 0.193. CI-2 is a member of the potato inhibitor 1 family; it is a serine proteinase inhibitor lacking disulfide bonds. Comparison of the subtilisin molecule in this complex with the native subtilisin shows that the two molecules are very similar in structure. The inhibitor binds in a mode presumably resembling that of a true substrate, but it is not cleaved. This is in accord with the reported structures of other serine proteinaseinhibitor complexes. CI-2 consists of a four-stranded mixed parallel and antiparallel f3-sheet against which an a-helix packs to form a hydrophobic core. A wide loop crossover connection between parallel strands 2 and 3 of the ,B-sheet contains the reactive-site bond. The conformation of the four residues to either side of the reactive-site bond is similar to that of the analogous residues in the third domain of the turkey ovomucoid inhibitor (Kazal family); the overall polypeptide chain fold ofthese inhibitors and the location of the reactive site in the respective chains are different.

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Soybean trypsin inhibitor (Kunitz) and its complex with trypsin. Carbon-13 nuclear magnetic resonance studies of the reactive site arginine

Cited by 54 publications

References 61 publications

13C- and 1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors

13C- and 1H-NMR studies of oxyanion and tetrahedral intermediate stabilization by the serine proteinases: optimizing inhibitor warhead specificity and potency by studying the inhibition of the serine proteinases by peptide-derived chloromethane and glyoxal inhibitors

Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator

Crystal and molecular structure of chymotrypsin inhibitor 2 from barley seeds in complex with subtilisin Novo

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