The Hydrolytic Water Molecule in Trypsin, Revealed by Time-Resolved Laue Crystallography

Singer, Paul; Smalås, Arne O.; Carty, Robert P.; Mangel, Walter F.; Sweet, Robert M.

doi:10.1126/science.8430314

Cited by 97 publications

(42 citation statements)

References 9 publications

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“…This is often the case, for example, in the refinement of structures of proteins and corresponding mutants or complexes with small molecules (Eriksson et al, 1993) and is generally the case in experiments involving Laue diffraction (Singer et al, 1993). The idealized and real cases examined here indicate that difference refinement (Fermi et al, 1982) offers substantial improvement in errors compared with independent refinement of the two structures.…”

Section: Discussionmentioning

confidence: 88%

Difference refinement: obtaining differences between two related structures

Terwilliger¹,

Berendzen²

1995

Acta Cryst D

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There are many examples in macromolecular crystallography where interest focuses on the differences between a previously determined 'native' structure and a nearly isomorphous 'variant'. In such cases, a useful approach to atomic refinement of the variant structure is through weighted least-squares minimization of the residual between the observed and calculated differences in amplitudes of structure factors, a strategy first used in the refinement of deoxycobalt hemoglobin [Fermi, Perutz, Dickinson & Chien (1982). J. Mol. Biol. 155,[495][496][497][498][499][500][501][502][503][504][505] and termed 'difference refinement'. For cases in which the modeling errors for the native and variant structures are correlated, theoretical arguments indicate that difference refinement should lead to improved estimates of structural differences when compared with conventional independent refinement. Tests employing simulated peptide data sets and real data from a wild-type protein and a mutant show that difference refinement can substantially reduce errors in the differences between structures when compared with independent refinement. The algorithm is very easy to implement and does not increase the computational demands of refinement.

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Section: Discussionmentioning

confidence: 88%

Difference refinement: obtaining differences between two related structures

Terwilliger¹,

Berendzen²

1995

Acta Cryst D

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“…The location of water molecule W2 in the oxyanion hole seemed at first glance a bit exotic for steric reasons and in view of the commonly accepted direct stabilization of the carbonyl oxygen by the oxyanion hole. However, in the bovine trypsin, a water molecule has been found in the same position (29). Its proposed role was to protonate the carbonyl oxygen atom of the substrate, to take up the excess electrons and facilitate the shift of electrons toward the oxyanion hole (5).…”

Section: Discussionmentioning

confidence: 99%

“…In the other models (QC1 and QC3), W2 remains a water molecule with strong internal bonds and external H bridges of normal strength. This indicates that, after the nucleophilic attack of W1, W2 regains its original state and functions as the Henderson water is supposed to function (29). The QC3 model corresponds to the end-state of peptide cleavage, step 5 in Fig.…”

Section: Discussionmentioning

confidence: 99%

Trypsin Revisited

Schmidt

Jelsch

Østergaard

et al. 2003

Journal of Biological Chemistry

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A series of crystal structures of trypsin, containing either an autoproteolytic cleaved peptide fragment or a covalently bound inhibitor, were determined at atomic and ultra-high resolution and subjected to ab initio quantum chemical calculations and multipole refinement. Quantum chemical calculations reproduced the observed active site crystal structure with severe deviations from standard stereochemistry and indicated the protonation state of the catalytic residues. Multipole refinement directly revealed the charge distribution in the active site and proved the validity of the ab initio calculations. The combined results confirmed the catalytic function of the active site residues and the two water molecules acting as the nucleophile and the proton donor. The crystal structures represent snapshots from the reaction pathway, close to a tetrahedral intermediate. The de-acylation of trypsin then occurs in true S N 2 fashion.

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“…The location of the water molecule necessary to hydrolyze the acyl-enzyme intermediate in the second step of the reaction has been the subject of considerable debate (40,41), but the most plausible candidate appears to be the solvent molecule (S-25) identified by Radisky et al (13) in their structures of productive acyl-enzyme complexes, and in crystal structures of acylenzymes formed with porcine pancreatic elastase (12, 42) and …”

Section: High-resolution Reconstruction Of the Serine-protease Mechanmentioning

confidence: 99%

Structure of a serine protease poised to resynthesize a peptide bond

Zakharova

Horvath

Goldenberg

2009

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

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The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzymesubstrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.trypsin ͉ bovine pancreatic trypsin inhibitor ͉ enzyme mechanisms S erine proteases are found throughout all 3 domains of cellular life and function in a wide range of physiological processes, including digestion, protein maturation and turnover, hemostasis, and immune responses (1). Approximately 0.6% of human protein-encoding genes are predicted to specify serine proteases, and this family is even more prevalent in other organisms, notably the arthropods (2, 3). A large body of biochemical and structural data have established a 2-step mechanism for hydrolysis of peptide bonds by this class of proteases (4), as shown in Scheme 1.The first step of the reaction is a nucleophilic attack by the catalytic serine residue (Ser-195 in trypsin and other members of the chymotrypsin, or S1, family) on the carbonyl carbon atom of the residue labeled P1, generating a covalent acyl-enzyme intermediate and a new peptide amino terminus, on the P1Ј residue. A second nucleophilic attack, by a water molecule, leads to hydrolysis of the acyl-enzyme, releasing the new carboxyl group and restoring the catalytic Ser residue to its initial state.

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The Hydrolytic Water Molecule in Trypsin, Revealed by Time-Resolved Laue Crystallography

Cited by 97 publications

References 9 publications

Difference refinement: obtaining differences between two related structures

Difference refinement: obtaining differences between two related structures

Trypsin Revisited

Structure of a serine protease poised to resynthesize a peptide bond

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