Structure of a specific acyl-enzyme complex formed between β-casomorphin-7 and porcine pancreatic elastase

Wilmouth, R.C.; Clifton, I.J.; Robinson, Carol V.; Roach, Peter L.; Aplin, Robin T.; Westwood, Nicholas J.; Hajdu, János; Schofield, Christopher J.

doi:10.1038/nsb0697-456

Cited by 69 publications

(84 citation statements)

References 32 publications

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“…Interestingly, the evolving carbanion could approach the acylated cysteine with a Buergi-Dunitz trajectory of 110° (Fig. 7C), which is within the expected range of 100 -110°for the transition state attack vector angle of a carbonyl group by a nucleophile (Nu 3 CϭO) (51,52). For similar stereoelectronic reasons, the carbanion should attack the Cys-171 thioester carbonyl with an obtuse angle as assumed for the related aldol reactions (53).…”

Section: Discussionsupporting

confidence: 62%

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

Schiebel¹,

Kapilashrami

Fekete

et al. 2013

Journal of Biological Chemistry

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Section: Discussionsupporting

confidence: 62%

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

Schiebel¹,

Kapilashrami

Fekete

et al. 2013

Journal of Biological Chemistry

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“…Thus, the role of HC2 in the reaction mechanism requires further investigations. The HC⅐TSG-6 intermediate has similarity with the acyl-enzyme intermediate of the serine proteases in which there is a transient covalent ester bond between the active site Ser 195 residue (chymotrypsin numbering system) and the C-terminal carbon of the substrate (42)(43)(44). In the serine proteases, an activated water molecule quickly hydrolyzes the ester bond to regenerate the enzyme.…”

Section: Discussionmentioning

confidence: 99%

TSG-6 Transfers Proteins between Glycosaminoglycans via a Ser28-mediated Covalent Catalytic Mechanism

Sanggaard

Sonne-Schmidt

Krogager

et al. 2008

Journal of Biological Chemistry

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Studies of the interaction between Bikunin proteins, tumor necrosis factor-stimulated gene-6 protein (TSG-6), and glycosaminoglycans have revealed a unique catalytic activity where TSG-6/heavy chain 2 transfer heavy chain subunits between glycosaminoglycan chains. The activity is mediated by TSG-6/ heavy chain 2 and involves a transient SDS stable interaction between TSG-6 and the heavy chain to be transferred. The focus of this study was to characterize the molecular structure of this cross-link to gain further insight into the catalytic mechanism. The result showed that the C-terminal Asp residue of the heavy chains forms an ester bond to Ser 28 ␤-carbon of TSG-6 suggesting that this residue plays a role during catalysis.

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“…A number of structures of acyl-enzymes with peptide substrates have been reported; however, the acyl-enzyme has generally been trapped by the use of a nonphysiologic low pH, by the use of a poor substrate with an exceptionally long-lived acyl-enzyme, or both (12,28,29). Such structures are necessarily imperfect approximations of the true intermediate in the cleavage of a favorable substrate.…”

Section: Discussionmentioning

confidence: 99%

“…A reaction-driven ''His flip'' mechanism has been proposed to address this question (4) but has met with resistance (5)(6)(7)(8). Another longstanding question concerns the positioning and activation of the hydrolytic water molecule and the trajectory of attack in the deacylation reaction (9)(10)(11)(12)(13).…”

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confidence: 99%

Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

Radisky

Lee

et al. 2006

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

124

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Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction.acyl-enzyme ͉ enzyme mechanism ͉ steady state ͉ reaction trajectory S erine proteases catalyze peptide bond hydrolysis in two sequential steps. In the first (acylation) reaction, the nucleophilic serine attacks the substrate scissile bond, forming first a tetrahedral intermediate and then a covalent acyl-enzyme with release of the C-terminal fragment. In the second (deacylation) reaction, a water molecule attacks the acyl-enzyme, leading to a second tetrahedral intermediate followed by release of the N-terminal fragment. This mechanism has long served as one of the classic paradigms illustrating the catalytic power of enzymes (1), yet crucial details remain controversial.It is generally accepted that a histidine residue acts as a general base in accepting a proton to activate serine as a nucleophile, and subsequently acts as a general acid, donating the proton to the nitrogen of the peptide leaving group (1). This same histidine is also presumed to deprotonate the hydrolytic water. What is not clear is why, if the histidine is ideally situated to deprotonate serine, does the first tetrahedral intermediate not collapse back to the reactant complex with return of the proton to serine? Does the active site undergo spatial reorganization as catalysis proceeds to favor the forward progress of the reaction (2, 3)? A reaction-driven ''His flip'' mechanism has been proposed to address this question (4) but has met with resistance (5-8).Another longstanding question concerns the positioning and activation of the hydrolytic water molecule and the trajectory of attack in the deacylation reaction (9-13).These questions can now be approached through the study of protein structures of intermediates along the catalytic pathway. Structures of the enzyme͞substrate Michaelis complex, analogs of the tetrahedral intermediates, and acyl-enzymes can yield great insight into the atomic motions and shifting geometric relationships along the reaction coordinate. Here, we report (i) high resolution structures of trypsin acylated by two good peptide substrates and (ii) higher resolution structures of two previously solved trypsin complexes: the stable acyl-enzyme formed with the poor substrate p-nitroguanidinobenzoate (14) and the covalent complex formed with the inhibitor leupeptin, which mimics a tetrahedral intermediate (15). Comparisons provide insight into active site adjustments involved in catalysis and cl...

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Structure of a specific acyl-enzyme complex formed between β-casomorphin-7 and porcine pancreatic elastase

Cited by 69 publications

References 32 publications

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

TSG-6 Transfers Proteins between Glycosaminoglycans via a Ser28-mediated Covalent Catalytic Mechanism

Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

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