Episelection: Novel Ki .apprx. Nanomolar Inhibitors of Serine Proteases Selected by Binding or Chemistry on an Enzyme Surface

Katz, B.A.; Finer-Moore, J.; Mortezaei, Reza; Rich, Daniel H.; Stroud, Rhonda M.

doi:10.1021/bi00026a008

Cited by 97 publications

(78 citation statements)

References 69 publications

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“…Fig. 2C shows a superposition of the catalytic residues of 4 structures: the BPTI-trypsin complex (carbon atoms colored green), the BPTI*-S195A trypsin complex (orange carbon atoms), bovine trypsin bound to a tetrahedral transition-state analog (purple carbon atoms) (16), and an acyl-enzyme intermediate formed by bovine trypsin and a peptide-nitroanilide substrate (gray carbon atoms) (13). The close superposition of the catalytic residues in the 4 structures suggests that the reaction proceeds with minimal structural changes in the active site.…”

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

confidence: 99%

“…Crystallographic studies of serine proteases with bound substrates and inhibitors have provided detailed structural information about the enzyme-substrate complex (8,9), the acylenzyme (10)(11)(12)(13), and the high-energy tetrahedral intermediates that separate these species (14)(15)(16)(17). Crystal structures with bound P1 products have also been reported (18)(19)(20), but at relatively low resolution or in forms that appear to be mixtures of the carboxyl, tetrahedral, and acyl-enzyme species.…”

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confidence: 99%

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

confidence: 99%

mentioning

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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“…Hence, some enzymes may undergo little conformational rearrangement while performing their functions, exemplified by the serine proteases. 1 Others may require surface loop movements to sequester the reactants from solution or position catalytic groups at the completion of the substrate binding step, as exemplified by L-lactate dehydrogenase where closure of the active site loop performs both of these tasks. 2 The next level of structural reorganization involves whole protein domain movements, and these are involved in a wide variety of functional roles.…”

Section: Introductionmentioning

confidence: 99%

“…However, until very recently all crystal structures known (from Bacillus stearothermophilus, horse, pig, and yeast) were of an open conformation of the protein such that the substrate and cofactor sites are too far apart to allow the transfer of phosphate from ATP to 3-PGA to give ADP and 1-3,bisphosphoglycerate (1,. Several experimental approaches to determine whether domain closure occurs during the catalytic reaction have been described, with somewhat contradictory results.…”

Section: Introductionmentioning

confidence: 99%

A general method of domain closure is applied to phosphoglycerate kinase and the result compared with the crystal structure of a closed conformation of the enzyme

et al. 1998

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The occurrence of large domain motions associated with the mechanism of action of many proteins is well established. We present a general method of predicting domain closure applicable to proteins containing domains separated by an apparent hinge. The method attempts to allow for natural directional bias within the closing protein by repeatedly applying a weak pulling force over a short distance between pairs of atoms chosen at random in the two domains in question. Appropriate parameters governing the pulling function were determined empirically. The method was applied to the bi-lobal protein PGK and a closed-form activated ternary complex generated for Bacillus stearothermophilus PGK. This model was compared with the recently determined crystal structure of closed-form Trypanosoma brucei PGK. The model predicts the correct hinge regions, although the magnitude of movement at one hinge point was overestimated, and provides a reasonable representation of the closed-form ternary complex.

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“…[10][11][12][13][14] We postulated that fragments bound to adjacent pockets within the active site of a protein could, in principle, self-assemble to generate larger, more potent ligands if they had complimentary chemical reactivities. [15] Such a method would have key advantages, as the time-consuming and expensive practice of conventional synthesis, purification, and testing of combinatorial libraries designed to discover potent inhibitors might be obviated.…”

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confidence: 99%