Active and Inactive Forms of the Transition-state Analog Protease Inhibitor Leupeptin: Explanation of the Observed Slow Binding of Leupeptin to Cathepsin B and Papain

Schultz, Richard M.; Varma-Nelson, P; Ortiz, Rafael; Kozlowski, K.A.; Orawski, Arthur T.; Pagast, Peter; Frankfater, Allen

doi:10.1016/s0021-9258(18)94215-8

Cited by 53 publications

(23 citation statements)

References 43 publications

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“…With peptidyl aldehyde inhibitors of serine and thiol proteases, the free aldehyde, not the hydrate, is the reactive inhibitory species (Schultz et al, 1989). If the free trifluoromethyl ketone is the species which reacts with chymotrypsin, then the observed rate constant (&on) might decrease at high enzyme concentration due to depletion of the ketone species.…”

Section: Resultsmentioning

confidence: 99%

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

Brady

Abeles²

1990

Biochemistry

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A series of seven peptidyl trifluoromethyl ketone (TFK) inhibitors of chymotrypsin have been prepared which differ at the P1 and P2 subsites. Inhibition equilibria and kinetics of association and dissociation with chymotrypsin have been measured. The association rate of Ac-Phe-CF3 was measured at enzyme concentrations between 8 nM and 117 microM in order to examine the relation between the ketone/hydrate equilibrium of trifluoromethyl ketones and the "slow binding" by these inhibitors. The association rate decreases at high enzyme concentrations, indicating that TFK ketone is the reactive species and that conversion of TFK hydrate to ketone becomes rate limiting under these conditions. Inhibitors with hydrophobic side chains at P2 bind more tightly but more slowly to chymotrypsin, indicating that formation of van der Waals contacts between the P2 side chain and the His 57 and Ile 99 side chains of chymotrypsin is a relatively slow process. Inhibitor properties were compared to the Michaelis-Menten kinetic constants of a homologous series of peptide methyl ester and peptide amide substrates. Plots of log Ki vs log (kcat/Km) are linear with slopes of 0.65 +/- 0.2, indicating that these inhibitors are able to utilize 65% of the total binding energy between chymotrypsin and its hydrolytic transition state.

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Section: Resultsmentioning

confidence: 99%

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

Brady

Abeles²

1990

Biochemistry

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“…The latter drawback might be partially explained by studies with the related protease inhibitor leupeptin (acetyl-Leu-Leu-Arg-H), which exists in solution mainly in its cyclized hemiaminal form (56%) and as a hydrate (42%), whereas only 2% of the inhibitory active aldehyde form was detected. Although equilibria between these three forms exist, the conversion of the hemiaminal and hydrate into the aldehyde species in water occurs with half-lives of approximately 7.5 and 1 min, respectively, which may limit their in vivo efficacy …”

Section: Synthetic Active Site-directed Inhibitorsmentioning

confidence: 99%

“…Although equilibria between these three forms exist, the conversion of the hemiaminal and hydrate into the aldehyde species in water occurs with half-lives of approximately 7.5 and 1 min, respectively, which may limit their in vivo efficacy. 146 3.7. Inhibitors with C-Terminal 4-Amidinobenzylamide.…”

Section: Synthetic Active Site-directed Inhibitorsmentioning

confidence: 99%

Fibrinolysis Inhibitors: Potential Drugs for the Treatment and Prevention of Bleeding

et al. 2019

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Hyperfibrinolytic situations can lead to lifethreatening bleeding, especially during cardiac surgery. The approved antifibrinolytic agents such as tranexamic acid, εaminocaproic acid, 4-aminomethylbenzoic acid, and aprotinin were developed in the 1960s without the structural insight of their respective targets. Crystal structures of the main antifibrinolytic targets, the lysine binding sites on plasminogen's kringle domains, and plasmin's serine protease domain greatly contributed to the structure-based drug design of novel inhibitor classes. Two series of ligands targeting the lysine binding sites have been recently described, which are more potent than the most-widely used antifibrinolytic agent, tranexamic acid. Furthermore, four types of promising active site inhibitors of plasmin have been developed: tranexamic acid conjugates targeting the S1 pocket and primed sites, substrate-analogue linear homopiperidylalanine-containing 4amidinobenzylamide derivatives, macrocyclic inhibitors addressing nonprimed binding regions, and bicyclic 14-mer SFTI-1 analogues blocking both, primed and nonprimed binding sites of plasmin. Furthermore, several allosteric plasmin inhibitors based on heparin mimetics have been developed.

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“…A similar process may account for the slow association rates of statine-based inhibitors to the aspartyl protease renin (Kati et al, 1987). The rate of association of the peptidyl aldehyde leupeptin to papain (Schultz et al, 1989) correlates with the kinetics of ring opening of an intramolecular carbinolamine isomer of leupeptin.…”

mentioning

confidence: 93%

Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors

Brady¹,

Wei²,

Ringe³

et al. 1990

Biochemistry

124

119

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The peptidyl trifluoromethyl ketones Ac-Phe-CF3 (1) and Ac-Leu-Phe-CF3 (2) are inhibitors of chymotrypsin. They differ in Ki (20 and 2 microM, respectively) as well as in their kinetics of association with chymotrypsin in that 1 is rapidly equilibrating, with an association rate too fast to be observed by steady-state techniques, while 2 is "slow binding", as defined by Morrison and Walsh [Morrison, J. F., & Walsh, C. T. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 202], with a second-order association rate constant of 750 M-1 s-1 at pH 7.0 [Imperiali, B., & Abeles, R. (1986) Biochemistry 25, 3760]. The crystallographic structures of the complexes of gamma-chymotrypsin with inhibitors 1 and 2 have been determined in order to establish whether structural or conformational differences can be found which account for different kinetic and thermodynamic properties of the two inhibitors. In both complexes, the active-site Ser 195 hydroxyl forms a covalent hemiketal adduct with the trifluoromethyl ketone moiety of the inhibitor. In both complexes, the trifluoromethyl group is partially immobilized, but differences are observed in the degree of interaction of fluorine atoms with the active-site His 57 imidazole ring, with amide nitrogen NH 193, and with other portions of the inhibitor molecule. The enhanced potency of Ac-Leu-Phe-CF3 relative to Ac-Phe-CF3 is accounted for by van der Waals interactions of the leucine side chain of the inhibitor with His 57 and Ile 99 side chains and by a hydrogen bond of the acetyl terminus with amide NH 216 of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

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Active and Inactive Forms of the Transition-state Analog Protease Inhibitor Leupeptin: Explanation of the Observed Slow Binding of Leupeptin to Cathepsin B and Papain

Cited by 53 publications

References 43 publications

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

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

Fibrinolysis Inhibitors: Potential Drugs for the Treatment and Prevention of Bleeding

Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors

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