Inhibition of chymotrypsin by peptidyl trifluoromethyl ketones: determinants of slow-binding kinetics

Brady, Kenneth D.; Abeles, Robert H.

doi:10.1021/bi00485a010

Cited by 72 publications

(94 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This conclusion is consistent with the relative inefficiency of cleavage at the tertiary and quaternary sites in wild-type Hap, where the P1 residue is phenylalanine. In chymotrypsin, proteolysis is also influenced by interaction between the P2 residue and the S2 subsite pocket (3). In this context, it is noteworthy that our work supports an important role for the P2 residue in Hap cleavage specificity, with leucine or glutamic acid favored over other amino acids.…”

Section: Discussionsupporting

confidence: 66%

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter

Kenjale¹,

Meng

Fink

et al. 2009

Infect Immun

View full text Add to dashboard Cite

Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the Hap S passenger domain, leaving the Hap ␤ C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.

show abstract

Section: Discussionsupporting

confidence: 66%

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter

Kenjale¹,

Meng

Fink

et al. 2009

Infect Immun

View full text Add to dashboard Cite

show abstract

“…If the inhibitor is still in the gem-diol form as shown in step B, which is the hypothesized mechanism, the inhibitor then needs to undergo a dehydration step to the ketone in step C. This step is most likely the rate limiting step of the reaction, which would agree with data published by Brady and Abeles for chymotrypsin inhibition. 33 This mechanism would also explain the time-dependence of both the kinetic and IC 50 data observed in this study as well as others. 27,29 For inhibitors such as the sulfoxide and sulfone, which demonstrate significantly slower binding kinetics (i.e, k i values) relative to the thioether, the dehydration reaction is the key step.…”

Section: Kinetic Studiessupporting

confidence: 80%

“…The main outstanding issues revolve around the hydration state of the "active" form of the inhibitor and the secondary interactions that occur within the active site of the enzyme that can affect inhibitor binding (mainly van der Waals interactions and electronic interactions such as pi-stacking). The issue of the geometry of the TFK in its active form (i.e., inhibitory form) has not been directly addressed in CaEs, but it has been shown for capthepsin B, 32 acetylcholinesterase 20 and chymotrypsin 33 that the ketone is the inhibitory species. These findings were further supported for CaEs by Roe et al, who postulated that the ketone is the inhibitory species for juvenile hormone esterase (JHE) inhibition, 34 which has been supported by 3-D QSAR studies 26 .…”

Section: Discussionmentioning

confidence: 99%

“…Work performed by the group of Abeles on the active form of peptide-based TFK inhibitors would most likely be an appropriate model. 20,33 In addition, the potential role of the putative intramolecular hydrogen bond needs to be addressed in greater detail. To date, most studies have attempted to infer the presence and/or role of the bond in inhibitor potency via ab initio calculations 29,30 or crystallography 29,51 .…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH)

Wheelock

Nishi

Ying

et al. 2008

Bioorganic & Medicinal Chemistry

View full text Add to dashboard Cite

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC 50 ) and binding kinetics (k i ). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1 H NMR, X-ray crystallography and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (~5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process.

show abstract

“…TMTFA was shown to behave as a tight binding time-dependent inhibitor, a behavior characteristic of other trifluoroketone inhibitors of serine proteases (30,31). In the recently published x-ray structure of TcAChE-TMTFA adduct (3), the bound ligand is thought to provide a constrained analog of ACh tetrahedral intermediate, with the oxyanion projecting toward the NH functions of the oxyanion hole.…”

Section: Modification Of Huache Hydrolytic Activity-replacementmentioning

confidence: 99%

Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase

Ordentlich¹,

Barak²,

Kronman³

et al. 1998

Journal of Biological Chemistry

148

View full text Add to dashboard Cite

The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analog m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000 -11,000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000 -6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500 -460-fold) differed from that toward the phosphates (12-95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly-122 seem to result from steric hindrance at the active center acyl pocket.

show abstract

Inhibition of chymotrypsin by peptidyl trifluoromethyl ketones: determinants of slow-binding kinetics

Cited by 72 publications

References 39 publications

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter

Influence of sulfur oxidation state and steric bulk upon trifluoromethyl ketone (TFK) binding kinetics to carboxylesterases and fatty acid amide hydrolase (FAAH)

Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase

Contact Info

Product

Resources

About