Inhibition of Elastase by <i>N</i>-Sulfonylaryl β-Lactams:  Anatomy of a Stable Acyl−Enzyme Complex<sup>,</sup>

Wilmouth, R.C.; Westwood, Nicholas J.; Anderson, K. G.; Brownlee, W. C.; Claridge, Timothy D. W.; Clifton, I.J.; Pritchard, Gareth J.; Aplin, Robin T.; Schofield, Christopher J.

doi:10.1021/bi9816249

Cited by 41 publications

(47 citation statements)

References 29 publications

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“…In general, our resulting model superposes very well with the 1.6 A Ê PPE model of an enzyme±inhibitor complex soaked with 20% glycerol (Wilmouth et al, 1998) and the 1.65 A Ê model of native PPE in 70% methanol (Meyer et al, 1988). Compared with the starting model of Wilmouth et al (1998), our structure has an r.m.s. deviation of 0.43 A Ê for all protein atoms and 0.14 A Ê for the C atoms.…”

Section: Short Communicationssupporting

confidence: 60%

“…Based on a cutoff radius of 1 A Ê , 85% of all water molecules in the model of Wilmouth et al (1998) and 78% of all water molecules in the model of Meyer et al (1988) can be considered as conserved in our model. This is especially valid for the internal water clusters of PPE as described by Meyer et al (1988).…”

Section: Short Communicationsmentioning

confidence: 99%

“…Data were processed using DENZO and SCALEPACK (Otwinowski & Minor, 1997 Navaza, 1994) using the 1.6 A Ê resolution structure of PPE (Wilmouth et al, 1998;PDB entry code 1btu). No signi®cant non-isomorphism was detected compared with the published structure.…”

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confidence: 99%

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Atomic resolution structure of native porcine pancreatic elastase at 1.1 Å

Würtele

Hahn

Hilpert

et al. 2000

Acta Crystallogr D Biol Crystallogr

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A data set from the serine protease porcine pancreatic elastase was collected at atomic resolution (1.1 A Ê) with synchrotron radiation. The improved resolution allows the determination of atom positions with high accuracy, as well as the localization of H atoms. Three residues could be modelled in alternative positions. The catalytic triad of elastase consists of His57, Asp102 and Ser195. The His57 N 1 H atom was located at a distance of 0.82 A Ê from the N 1 atom. The distance between His57 N 1 and Asp102 O 2 is 2.70 AE 0.04 A Ê , thus indicating normal hydrogen-bonding geometry. Additional H atoms at His57 N 42 and Ser195 O could not be identi®ed in the F o À F c density maps.

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Section: Short Communicationssupporting

confidence: 60%

Section: Short Communicationsmentioning

confidence: 99%

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Atomic resolution structure of native porcine pancreatic elastase at 1.1 Å

Würtele

Hahn

Hilpert

et al. 2000

Acta Crystallogr D Biol Crystallogr

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“…the slow deacylation rates observed with certain stable acyl-enzyme complexes (44,45)), the current structures (Fig. 6) support an optimal mechanism for efficient serine protease catalysis in which every step occurs in a stereoelectronically favored manner and in which there is minimal movement of the histidine residue that enables general acid/base catalysis.…”

Section: Discussionsupporting

confidence: 64%

Structural Analyses on Intermediates in Serine Protease Catalysis

Liu

Schofield

Wilmouth

2006

Journal of Biological Chemistry

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Although the subject of many studies, detailed structural information on aspects of the catalytic cycle of serine proteases is lacking. Crystallographic analyses were performed in which an acyl-enzyme complex, formed from elastase and a peptide, was reacted with a series of nucleophilic dipeptides. Multiple analyses led to electron density maps consistent with the formation of a tetrahedral species. In certain cases, apparent peptide bond formation at the active site was observed, and the electron density maps suggested production of a cis-amide rather than a trans-amide. Evidence for a cis-amide configuration was also observed in the noncovalent complex between elastase and an ␣ 1 -antitrypsin-derived tetrapeptide. Although there are caveats on the relevance of the crystallographic data to solution catalysis, the results enable detailed proposals for the pathway of the acylation step to be made. At least in some cases, it is proposed that the alcohol of Ser-195 may preferentially attack the carbonyl of the cis-amide form of the substrate, in a stereoelectronically favored manner, to give a tetrahedral oxyanion intermediate, which undergoes N-inversion and/or C-N bond rotation to enable protonation of the leaving group nitrogen. The mechanistic proposals may have consequences for protease inhibition, in particular for the design of high energy intermediate analogues.Following from their role in the development of mechanistic and structural enzymology, serine proteases, and more generally enzymes catalyzing hydrolysis or related reactions proceeding via an acyl-enzyme complex, have been shown to be of importance in human diseases. Serine proteases, including thrombin, factor X, and elastase, are targets for medicinal chemistry (1, 2). One important class of inhibitors is designed to mimic a tetrahedral intermediate, but until recently there has been little three-dimensional structural information on the early intermediates in catalysis.In outline, the accepted mechanism for serine proteases involves nucleophilic attack by the alcohol of Ser-195 onto the amide carbonyl of the peptide substrate to form a first tetrahedral intermediate. This intermediate collapses to form an ester, or acyl-enzyme complex, and the COOH-terminal product fragment then leaves the active site. Hydrolysis of the Ser-195-linked ester bond then occurs via a second tetrahedral intermediate, to form the NH 2 -terminal product fragment and return the active site to its resting state. Much evidence has accumulated in support of this general mechanism, including the role of His-57 in enabling acid/base catalysis, and it has been extended to other types of hydrolysis reaction, including esterases. Although there has been extensive structural work on covalently bound inhibitors and some on substrate analogues (3), there is relatively little direct crystallographic evidence for the key intermediates, and questions remain as to the precise stereoelectronic pathway of the reaction, including the timing and nature of the requisite proton transfers (4)...

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“…This suggests either WAT2 or WAT3 as the deacylating water, with the long lived stability of the penem inhibitor acyl-enzyme complex a result of displacement of the deacylating water. Inhibitors for TEM-1 ␤-lactamase have been designed based on this principle (34,35), and analysis of serine protease acyl-enzyme complexes have revealed similar effects (36,37).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of a Bacterial Signal Peptidase Apoenzyme

Paetzel¹,

Dalbey²,

Strynadka³

2002

Journal of Biological Chemistry

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We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(⌬2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 Å resolution in a different space group (P4 1 2 1 2) from that of the previously published acyl-enzyme inhibitorbound structure (P2 1 2 1 2) (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186 -190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 O␥H) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. Type I signal (leader) peptidase (SPase, 1 EC 3.4.21.89) is the membrane-bound serine endoprotease responsible for cleavage of the amino-terminal signal (or leader) peptide extensions from secretory proteins and some membrane proteins (for recent reviews see Refs. 1 and 2). The type I SPase from Escherichia coli has served as the model Gram-negative SPase and is the most thoroughly characterized SPase to date. It has been cloned (3), sequenced (4), overexpressed (5), purified (4, 6, 7), and kinetically (8) and structurally (9) characterized. Sitedirected mutagenesis (8, 10) and chemical modification (8,11) studies are consistent with SPase utilizing a Ser-Lys dyad mechanism, whereby Ser-90 serves as the nucleophile and Lys-145 serves as the general base. E. coli SPase (323 amino acids, 35,988 Da, pI 6.9) contains two amino-terminal transmembrane segments (residues 4 -28 and 58 -76), a small cytoplasmic region (residues 29 -58), and a carboxyl-terminal periplasmic catalytic region (residues 77-323). A catalytically active fragment of SPase (SPase-(⌬2-75)), which lacks the two transmembrane segments and the cytoplasmic domain, has been cloned, overexpressed, characterized (12, 13), and crystallized (14). The ⌬2-75 construct required detergent or lipid for optimal activity (13) and crystallization (14). The crystal structure of SPase-(⌬2-75) in complex with a covalently bound ␤-lactambased inhibitor revealed directly that Ser-90 was the nucleophile and that the position of the N of Lys-145 was consistent with a role as the general base (9) in the catalytic mechanism. The shallow hydrophobic pockets observed adjacent to the catalytic residues helped to explain the Ala-X-Ala substrate specificity. The structure also revealed that SPase is an unusual serine protease in that it attacks the substrate scissile amide carbonyl from the si-face rather than the re-face as seen in most serine proteases (15,16).An important component of the catalytic ...

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Inhibition of Elastase by N-Sulfonylaryl β-Lactams: Anatomy of a Stable Acyl−Enzyme Complex^,

Cited by 41 publications

References 29 publications

Atomic resolution structure of native porcine pancreatic elastase at 1.1 Å

Atomic resolution structure of native porcine pancreatic elastase at 1.1 Å

Structural Analyses on Intermediates in Serine Protease Catalysis

Crystal Structure of a Bacterial Signal Peptidase Apoenzyme

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Inhibition of Elastase by N-Sulfonylaryl β-Lactams: Anatomy of a Stable Acyl−Enzyme Complex,

Cited by 41 publications

References 29 publications

Atomic resolution structure of native porcine pancreatic elastase at 1.1 Å

Atomic resolution structure of native porcine pancreatic elastase at 1.1 Å

Structural Analyses on Intermediates in Serine Protease Catalysis

Crystal Structure of a Bacterial Signal Peptidase Apoenzyme

Contact Info

Product

Resources

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Inhibition of Elastase by N-Sulfonylaryl β-Lactams: Anatomy of a Stable Acyl−Enzyme Complex^,