Mechanism of association of N-acetyl-L-phenylalanylglycinal to papain

Frankfater, Allen; Kuppy, Thresiamma

doi:10.1021/bi00522a026

Cited by 38 publications

(47 citation statements)

References 27 publications

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“…This is further supported by our previous theoretical studies of thiolate reactivity towards different electrophiles,30 and the conclusions derived from experimental kinetic studies of papain 31. We also demonstrated that with a weak Cys nucleophile in Cathepsin K, the binding trend of CS of RCA inhibitors is dominated by the change in pK a of the oxygen atom in TC(OH), which is accounted for by the covalent descriptor W2 13.…”

Section: Resultssupporting

confidence: 84%

EMBM − A New Enzyme Mechanism-Based Method for Rational Design of Chemical Sites of Covalent Inhibitors

Traube

Vijayakumar

Hirsch

et al. 2010

J. Chem. Inf. Model.

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We introduce an enzyme mechanism-based method, EMBM, aimed at rational design of chemical sites, CS, of reaction coordinate analog inhibitors. The energy of valence reorganization of CS, caused by the formation of the enzyme-inhibitor covalent complex, is accounted for by new covalent descriptors W1 and W2. We considered CS fragments with a carbonyl reactivity center, like in native protease substrates. The W1 and W2 descriptors are calculated quantum mechanically on small molecular clusters simulating the reaction core of the formed covalent tetrahedral complex -anionic TC(O−) or neutral TC(OH). The modeling on a reaction core allows generation of various CS and corresponding TC(O−) and TC(OH) as universal building blocks of real inhibitors and their covalent complexes with serine or cysteine hydrolases. Moreover, the approach avoids the need for 3D structure of the target enzyme, so EMBM may be used for ligand-based design. We have built a Chemical Site of Inhibitors (CSI) databank with pairs of W1 and W2 descriptors pre-calculated for both CH 3 O(−) and CH 3 S(−) nucleophiles for every collected CS fragment. We demonstrated that contribution of a CS fragment to the binding affinity of an inhibitor depends on both its covalent reorganization during the chemical transformation and its non-covalent interactions in the enzyme active site. Consequently, prediction of inhibitors binding trend can be done only by accounting for all of these factors, using W1 and W2 in combination with non-covalent QSAR descriptors.

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Section: Resultssupporting

confidence: 84%

EMBM − A New Enzyme Mechanism-Based Method for Rational Design of Chemical Sites of Covalent Inhibitors

Traube

Vijayakumar

Hirsch

et al. 2010

J. Chem. Inf. Model.

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“…This therefore lends support to the interpretation of our structure as a tetrahedral adduct. Frankfater et al [30] proposed a three-step pathway for the inhibition of papain by an aldehyde inhibitor in which two tetrahedral adducts are generated in succession. In the first adduct the oxygen is in the oxyanion hole, whereas in the second it is not.…”

Section: Discussionmentioning

confidence: 99%

X‐ray crystallographic structure of a papain‐leupeptin complex

et al. 1993

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The three‐dimensional structure of the papain‐leupeptin complex has been determined by X‐ray crystallography to a resolution of 2.1 Å (overall R‐factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the S'subsites; (ii) the ‘carbonyl’ carbon atom of the inhibitor is covalently bound by the Cys‐25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the ‘carbonyl’ oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln‐19 and Cys‐25.

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“…Transition-state analog inhibitors are predicted to have affinity to enzyme active sites orders of magnitude higher than even the natural substrates of the enzyme (31,32). In agreement with a mechanism of transition-state analog binding for peptidyl aldehyde analogs of specific proteinase substrates, the affinity constants found for peptidyl aldehyde associations are 102_105 times greater than those of analogous peptide substrates to the enzyme (20)(21)(22)(23)(24)(25)(26). In accord with this expectation, we found an equilibrium dissociation constant of 4.37 …”

Section: Resultsmentioning

confidence: 78%

“…1, in which RCH=O is the peptidyl aldehyde inhibitor, E-X is the active site nucleophile (serine -OH or cysteine YS-) of the respective enzyme, IA is the tetrahedral oxanion intermediate, and IIB is the stable tetrahedral hemiacetal or thiohemiacetal complex between aldehyde and enzyme (21,26). Structure TIB contains a tetrahedrally liganded carbonyl carbon (Sp3 electronic configuration) similar to the proposed structure of the reactive carbonyl carbon in the transition-state of peptide hydrolysis (20)(21)(22)(23)(24)(25)(26). Thus, the complex of peptidyl aldehydes with enzyme mimics the transition-state configuration in substrate hydrolysis, and DnsArgH is a transition-state analog inhibitor.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, both the neutral trypsin-like serine proteinases and the cathepsin-B-like cysteinyl proteinase, previously reported to be associated with transformed cells should be detected with DnsArgH. The fluorescent aldehyde binds to proteinases with a high affinity and selectivity because of the transition-state-like properties of the aldehyde proteinase association complex (20)(21)(22)(23)(24)(25)(26), and only on association into the enzyme active site does DnsArgH give a high fluorescent yield. We have used DnsArgH to both visualize and quantify the concentrations of cysteine and serine trypsin-like proteinases in fibrosarcoma cells and normal fibroblasts.…”

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confidence: 93%

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Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.

Kozlowski¹,

Wezeman²,

Schultz³

1984

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

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The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteinase inhibitor p-chloromercuribenzoate. Significant evidence exists for the role of proteolytic enzymes in malignant transformation, tumor cell invasion, and tumor cell metastasis (1-3). For example, treatment of normal cells with proteolytic enzymes can lead to the appearance of phenotypic characteristics in the untransformed cells that are similar to those of transformed cells (1, 4-7). In addition, proteolytic enzymes secreted from transformed cells may degrade host tissues into which the tumor cells invade, and may, through the degradation of surrounding tissue, promote tumor cell metastasis (1-3). Particular proteinases are reported to be secreted in larger amounts by transformed cells than their normal counterparts. These identified proteinases secreted by transformed cells include the serinetype proteinase plasminogen activator (1, 2, 8, 9), the lysosomal cysteine-type proteinase cathepsin-B (1, 10), and collagenolytic proteinases (1,3). The existence of a role for proteolytic enzymes in tumorigenesis and metastasis is supported by experiments in which these processes are shown to be inhibited by inhibitors of proteinase enzymes (1, 2).Whereas a large amount of evidence exists for secretion into the extracellular environment of relatively high concentrations of proteinases by tumor cells, recent reports also document the existence of proteinase activity either associated with the outer cell membrane or membrane fractions within transformed cells (1,2,(11)(12)(13)(14)(15)(16)(17)(18)(19). Similar to the types of proteinases reported to be found secreted into extracellular fluid, both a neutral trypsin-like seine proteinase, which may be plasminogen activator (1,2,(11)(12)(13)(14)(15)(16), and a cathepsin-B cysteine proteinase (1, 17) have been reported. A high concentration of cell surface proteinases may provide an intimate mechanism by which the membrane of the transformed cell can both maintain malignant traits within the tumor and promote the degradation of surrounding tissue during tumor cell invasion and metastasis.In this communication we report the development of a general proteinase fluorescent probe, dansyl-L-argininal (DnsArgH), which is capable of detecting both serine and cysteine proteinases with a primary site specificity toward arginine. Thus, both the neutral trypsin-like serine proteinases and the cathepsin-B-like cysteinyl proteinase, previously reported to be associated with transformed cells should be detected with DnsArgH. The fluorescent aldehyde binds to proteinases with a high affinity an...

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Mechanism of association of N-acetyl-L-phenylalanylglycinal to papain

Cited by 38 publications

References 27 publications

EMBM − A New Enzyme Mechanism-Based Method for Rational Design of Chemical Sites of Covalent Inhibitors

EMBM − A New Enzyme Mechanism-Based Method for Rational Design of Chemical Sites of Covalent Inhibitors

X‐ray crystallographic structure of a papain‐leupeptin complex

Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.

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