RESUMO
High molecular weight penicillin-binding proteins (PBPs) are bifunctional enzymes that build bacterial cell walls from the glycopeptide lipid II [GlcNAc-MurNAc(L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala)-pyrophosphate-undecaprenol] by a process involving disaccharide polymerization and peptide cross-linking. The latter reaction involves acyl-transfer chemistry in which the penultimate (D)Ala first acylates the active-site serine, with release of the terminal (D)Ala, and is then transferred to the epsilon-amine of a Lys on a neighboring pentapeptide chain. These enzymes also catalyze hydrolysis of specific thioester substrates and acylation by beta-lactam antibiotics. In this paper, we explore these latter two reactions and report mechanistic experiments on the reaction of Streptococcus pneumoniae PBP 2x with N-benzoyl-(D)Ala-thioacetic acid [Bz-(D)Ala-(S)Gly] and penicillin G. For these experiments, we used PBP 2x, a soluble form of PBP 2x in which the transmembrane domain was deleted. The following results are significant: (1) pH dependencies for acylation of PBP 2x by penicillin G and Bz-(D)Ala-(S)Gly are identical, suggesting that the same ionizable residues are involved in both reactions and that these residues play the same catalytic role in the two processes. On the basis of these results, we propose a mechanistic model that is also consistent with recently published structural data [Gordon, E., et al. (2000) J. Mol. Biol. 299, 477-485]. (2) Pre-steady-state experiments for the PBP 2x-catalyzed hydrolysis of Bz-(D)Ala-(S)Gly at pH 6.5 indicate that k(c) is principally rate-limited by acylation with some contribution from deacylation. The contribution of these steps to rate limitation is pH-dependent, with acylation entirely rate-limiting at pH values less than 5.5 and deacylation principally rate-limiting above pH 8.5. (3) Results of solvent isotope effect and proton inventory experiments for acylation suggest a complex process that is at least partially rate-limited by chemistry with some involvement of changes in solvation and/or enzyme conformation. (4) Analysis of activation parameters suggests that during the acylation of PBP 2x by penicillin G the inherent chemical stability of penicillin's amide bond, as manifested in the enthalpy of activation, is offset by a favorable entropy term that reflects penicillin's rotationally constrained bicyclic system, which presumably allows a less energetically demanding entry into the transition state for acylation.
Assuntos
Alanina/análogos & derivados , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Ligação às Penicilinas , Streptococcus pneumoniae/enzimologia , Acilação/efeitos dos fármacos , Alanina/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Catálise , Clonagem Molecular , Deutério/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Penicilina G/metabolismo , Penicilina G/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Prótons , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solventes , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Especificidade por Substrato , Temperatura , Termodinâmica , Tioglicolatos/metabolismoRESUMO
gamma-Glutamyl transpeptidase (gammaGTase) catalyzes the transfer of the gamma-glutamyl moiety of gamma-glutamyl-derived peptides, such as glutathione (gammaGlu-Cys-Gly), and anilides, such as gamma-glutamyl-7-amido-4-methylcoumarin (gammaGlu-AMC), to acceptor molecules, including water and various dipeptides. These acyl-transfer reactions all occur through a common acyl-enzyme intermediate formed from attack of an active site hydroxyl on the gamma-carbonyl carbon of gammaGlu-X with displacement of X. In this paper, we report that gammaGTase is potently inhibited by the gamma-boronic acid analogue of L-glutamic acid, 3-amino-3-carboxypropaneboronic acid (gamma-boroGlu). We propose that gamma-boroGlu adds to the active site hydroxyl of gammaGTase to form a covalent, tetrahedral adduct that resembles tetrahedral transition states and intermediates that occur along the reaction pathway for gammaGTase-catalyzed reactions. Our studies demonstrate that gamma-boroGlu is a competitive inhibitor of the gammaGTase-catalyzed hydrolysis of gammaGlu-AMC with a K(i) value of 35 nM. Kinetics of inhibition studies allow us to estimate the following values: k(on) = 400 mM(-1) s(-1) and k(off) = 0.02 s(-1). We also found that gamma-boroGlu is an uncompetitive inhibitor of Gly-Gly-promoted transamidation of gammaGlu-AMC. This observation is consistent with the kinetic mechanism we determined for gammaGTase-catalyzed transamidation of gammaGlu-AMC by Gly-Gly to form gammaGlu-Gly-Gly. To probe rate-limiting transition states for gammaGTase catalysis and inhibition, we determined solvent deuterium isotope effects. Solvent isotope effects on k(c)/K(m) for hydrolysis of gammaGlu-AMC and k(on) for inhibition by gamma-boroGlu are identical and equal unity, suggesting that the processes governed by these rate constants are both rate-limited by a step that is insensitive to solvent deuterium such as a conformational fluctuation of the initially formed E-S or E-I complex. In contrast, the solvent isotope effect on k(c) is 2.4. k(c) is rate-limited by hydrolysis of the acyl-enzyme intermediate that is formed during reaction of gammaGTase with gammaGlu-AMC. Thus, the magnitude of this isotope effect suggests the formation of a catalytically important protonic bridge in the rate-limiting transition state for deacylation.
Assuntos
Compostos de Boro/metabolismo , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/metabolismo , gama-Glutamiltransferase/antagonistas & inibidores , gama-Glutamiltransferase/metabolismo , Acilação , Amidas/metabolismo , Ligação Competitiva , Ácidos Borônicos/metabolismo , Catálise , Cumarínicos/metabolismo , Deutério , Relação Dose-Resposta a Droga , Glicina/metabolismo , Hidrólise , Cinética , SolventesRESUMO
Signal peptidases of prokaryotic organisms reside in the outer leaflet of the cytoplasmic membrane and catalyze the hydrolytic cleavage of a specific peptide bond of membrane-imbedded preproteins to liberate mature proteins for secretion. In this manuscript, we report new and efficient peptide substrates for SPase and their use to explore features of this enzyme's reaction mechanism. The enzyme used in this study was recombinant SPase I of Escherichia coli that had been solubilized with Triton X-100 and purified to near homogeneity. Our new substrates are based on the fluorogenic peptide reported by Zhong and Benkovic [(1998) Anal. Biochem. 255, 66], Y(NO2)FSASALA approximately KIK(Abz)-NH(2) (Y(NO2), 3-nitro-L-tyrosine; K(Abz), epsilon-(2-aminobenzoyl)-L-Lys; hydrolysis at A approximately K). We found that when a signal peptide-like sequence is appended onto the N-terminus of this peptide to produce K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2), k(c)/K(m) increases from 85 to 2.5 x 10(6) M(-)(1) s(-)(1). k(c)/K(m) decreases with increasing concentration of Triton X-100 micelles under the condition [Triton X-100](micelle) > [S](0) > [E](0). We explain this apparent inhibition with a model of surface dilution kinetics in which "empty" micelles compete with substrate-containing micelles for micelle-bound enzyme. Fusion of micelle-bound enzyme with a substrate-containing micelle leads to formation of productive E:S substrate complexes while fusion of micelle-bound enzyme with an "empty" micelle is nonproductive and inhibitory. The dependence of steady-state kinetic parameters for the SPase-catalyzed hydrolysis of K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2) on [Triton X-100](micelle) supports this model. Product inhibition and solvent isotope effects were also investigated and could be interpreted in the context of this model.
Assuntos
Escherichia coli/enzimologia , Proteínas de Membrana , Serina Endopeptidases/metabolismo , Sequência de Bases , Catálise , Primers do DNA , Hidrólise , Cinética , Octoxinol , Serina Endopeptidases/química , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Although there is strong epidemiologic evidence linking Schistosoma haematobium infection with carcinoma of the bladder, the utility of cytologic screening for urinary tract cancer has not been critically evaluated in S. haematobium-endemic populations. The present cross-sectional study examined urine cytology findings among 1,014 residents (ages 1 to 91) of the S. haematobium-endemic Msambweni area of Coast Province, Kenya. Among 705 evaluable cytology specimens, prevalence of inflammation (39%), hyperkeratosis (30%), metaplasia (33%), and frank atypia (0.4%) was notably higher than in previously studied, non-endemic populations. Overall, S. haematobium infection was strongly associated with increased risk for cytologic abnormality (> 2.8-fold relative risk of metaplasia or hyperkeratosis; P < 0.001). Age-group analysis confirmed parallel increases in metaplasia and S. haematobium infection prevalence early in life (from age I to 15 for both boys and girls). However, above age 20, metaplasia prevalence persisted at 33-45% prevalence despite a decline in infection prevalence and intensity. Prevalence of advanced (moderate or severe) metaplasia showed two age-related peaks: the first at 10-14 years of age (at the time of peak infection), and the second among subjects > or = 60 years old. No cancers were detected in the study population either on cytology or on follow-up ultrasound examination. These data suggest an age-dependent progression of cellular abnormalities in the urinary epithelium that is associated with chronic S. haematobium infection, which becomes independent of concurrent infection intensity as subjects grow older. Implications for cancer screening are discussed.
Assuntos
Carcinoma de Células Escamosas/epidemiologia , Carcinoma de Células Escamosas/etiologia , Esquistossomose Urinária/complicações , Neoplasias da Bexiga Urinária/epidemiologia , Neoplasias da Bexiga Urinária/etiologia , Sistema Urinário/patologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/patologia , Criança , Pré-Escolar , Estudos Transversais , Epitélio , Feminino , Humanos , Lactente , Quênia/epidemiologia , Masculino , Metaplasia , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Ultrassonografia , Neoplasias da Bexiga Urinária/patologia , Sistema Urinário/diagnóstico por imagem , Urina/citologiaRESUMO
Surveys have revealed that nonusers of hearing aids frequently hold negative perceptions of hearing aid users (the "hearing aid effect"). This study involved the use of both objective and subjective approaches in measuring the perceptions of older females toward hearing aid wearers. Twenty older females rated an aided and unaided peer after reading to them. Between-group differences were insignificant for objective measures (vocal intensity, reading time) and two subjective measures (age, attractiveness). However, the older females perceived their aided peers significantly more negatively than their unaided peers on measures of confidence, intelligence, and friendliness. This negative perception also surfaced in females who were unaware of the hearing aids, suggesting that a negative self-image by hearing aid wearers may contribute to the hearing aid effect.
Assuntos
Atitude , Auxiliares de Audição , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Feminino , Transtornos da Audição/terapia , Humanos , Pessoa de Meia-IdadeRESUMO
We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (lambda ex = 340 nm, lambda em = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 nM and 12 nM, respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition of the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities, dissociation constants varying by less than 25-fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are >300-fold more potent for inhibiting ChT-L activity than for PGPH activity. Inactivation of the BrAAP activity of the proteasome by clasto-lactacystin beta-lactone is also biphasic. beta-Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotrypsin-like activity. The remaining 40% of the BrAAP activity is inactivated by beta-lactone at a 50-fold slower rate, with kinetics indistinguishable from its inactivation of the PGPH activity. These results suggest a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e., BrAAP activity) occurs at two different active sites in the 20S proteasome, and that these two active sites are the same ones that catalyze the previously described ChT-L and PGPH activities.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Lactonas/farmacologia , Complexos Multienzimáticos/metabolismo , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Aldeídos/farmacologia , Animais , Inibidores de Cisteína Proteinase/farmacologia , Endopeptidases/efeitos dos fármacos , Cinética , Espectrometria de Massas , Complexo de Endopeptidases do Proteassoma , Coelhos , Especificidade por SubstratoRESUMO
Deubiquitinating enzymes constitute a family of cysteine hydrolases that specifically cleave ubiquitin-derived substrates of general structure Ub-X, where X can be any number of leaving groups ranging from small thiols and amines to Ub and other proteins (Ub, ubiquitin). We have developed a general assay for deubiquitinating enzymes based on the substrate ubiquitin C-terminal 7-amido-4-methylcoumarin (Ub-AMC). Ub-AMC is efficiently hydrolyzed with liberation of highly fluorescent AMC by two rabbit reticulocyte deubiquitinating enzymes: isopeptidase T (IPaseT), a member of the gene family of ubiquitin-specific processing enzymes, and UCH-L3, a member of the family of ubiquitin C-terminal hydrolases. We used this new assay to probe kinetic and mechanistic aspects of catalysis by IPaseT and UCH-L3. Results from four series of experiments are discussed: (1) For UCH-L3, we determined steady-state kinetic parameters that suggest a diffusion-limited reaction of UCH-L3 with Ub-AMC. To probe this, we determined the viscosity dependence of kc/Km, as well as kc. We found complex viscosity dependencies and interpreted these in the context of a model in which association and acylation are viscosity-dependent but deacylation is viscosity-independent. (2) The kinetics of inhibition of UCH-L3 by ubiquitin C-terminal aldehyde (Ub-H) were determined and reveal a Ki that is less than 10(-14) M. Several mechanisms are considered to account for the extreme inhibition. (3) The IPaseT-catalyzed hydrolysis of Ub-AMC is modulated by Ub with activation at low [Ub] and inhibition at high [Ub]. (4) Finally, we compare kc/Km values for deubiquitinating enzyme-catalyzed hydrolysis of Ub-AMC and Z-Leu-Arg-Gly-Gly-AMC. For IPaseT, the ratio of rate constants is 10(4), while for UCH-L3 this ratio is > 10(7). These results suggest the following: (i) Deubiquitinating enzymes are able to utilize the free energy that is released from remote interactions with Ub-containing substrates for stabilization of catalytic transition states, and (ii) UCHs are more efficient at utilizing the energy from these interactions, presumably because they do not possess a binding domain for a Ub "leaving group".
Assuntos
Cumarínicos/metabolismo , Endopeptidases/metabolismo , Corantes Fluorescentes/metabolismo , Oligopeptídeos/metabolismo , Ubiquitinas/metabolismo , Animais , Carbono-Nitrogênio Liases/metabolismo , Catálise , Bovinos , Hidrólise , Cinética , Coelhos , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/metabolismo , Ubiquitina Tiolesterase , Ubiquitinas/análogos & derivados , Ubiquitinas/farmacologia , ViscosidadeRESUMO
Potent and selective dipeptidyl boronic acid proteasome inhibitors are described. As compared to peptidyl aldehyde compounds, boronic acids in this series display dramatically enhanced potency. Compounds such as 15 are promising new therapeutics for treatment of cancer and inflammatory diseases.
Assuntos
Ácidos Borônicos/farmacologia , Cisteína Endopeptidases/efeitos dos fármacos , Complexos Multienzimáticos/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Ácidos Borônicos/química , Estrutura Molecular , Inibidores de Proteases/química , Complexo de Endopeptidases do ProteassomaRESUMO
The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Complexos Multienzimáticos/metabolismo , Acetilcisteína/química , Acetilcisteína/farmacologia , Transporte Biológico , Glutationa/química , Células HeLa , Humanos , Lactonas/farmacologia , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Células Tumorais CultivadasRESUMO
Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquitin (polyUb) chains, simple C-terminal derivatives of Ub, and certain peptides. We recently reported that IPaseT is regulated by ubiquitin (Ub); while submicromolar Ub activates, higher concentrations inhibit this enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain these observations, we proposed a model for IPaseT involving two binding sites for Ub. According to the model, the two sites are adjacent to one another and are the extended active site that binds two Ub moieties of a polyUb chain. The "activation site" binds the Ub that donates Lys to the isopeptide bond. The "inhibition site" is adjacent and binds the Ub that donates the C-terminal Gly to the isopeptide bond. We now report that the interaction of IPaseT with the C-terminal aldehyde of Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibits IPaseT with a Ki of 2.3 nM, while at 0.6 microM Ub, where the "activation site" is occupied, Ki is less than 0.1 nM. At high Ub concentrations, where both the "activation" and "inhibition" sites are occupied, IPaseT cannot bind Ub-H. We also determined the kinetics of inhibition of IPaseT by Ub-H. In the absence of Ub, a two-step mechanism is followed. In the first step, Ub-H slowly combines with IPaseT to form a relatively weak complex (K1 = 260 nM) that slowly isomerizes to the final, stable complex that accumulates in the steady-state (k2 = 2 x 10(-3) s-1; k-2 = 0.02 x 10(-3) s-1). In contrast, Ub-activated IPaseT is inhibited by Ub-H through a three-step process. In the first step, Ub-H rapidly combines with IPaseT to form a complex (K1 = 10 nM) that slowly isomerizes to a second, more stable complex (k2 = 18 x 10(-3) s-1; k-2 = 1.5 x 10(-3) s-1). In the third step, the second complex converts to the final complex (k3 = 1.5 x 10(-3) s-1; k-3 < 0.2 x 10(-3) s-1). To unify the results of this study with our previous results on catalysis, we propose that binding of Ub either to catalytic transition states or to tetrahedral inhibition intermediates liberates more free energy than binding of Ub to the reactant state of IPaseT and that IPaseT can utilize this binding energy to stabilize both of these tetrahedral species. The overall effect is a Ub-induced increase in catalytic efficiency or inhibitory potency.
Assuntos
Carbono-Nitrogênio Liases , Inibidores Enzimáticos/farmacologia , Liases/antagonistas & inibidores , Ubiquitinas/análogos & derivados , Animais , Hidrólise , Cinética , Liases/metabolismo , Peptídeos/metabolismo , Coelhos , Reticulócitos/enzimologia , Espectrometria de Fluorescência , Termodinâmica , Ubiquitinas/metabolismo , Ubiquitinas/farmacologiaRESUMO
In this paper, we report kinetic studies for the chymotryptic activity of the 20S proteasome. Major observations include the following: (1) Reaction progress curves that are recorded at concentrations of Suc-Leu-Leu-Val-Tyr-AMC greater than about 40 microM are biphasic and characterized by initial velocities that decay by a first-order process to final, steady-state velocities. (2) Also at [Suc-Leu-Leu-Val-Tyr-AMC] > 40 microM, initial and steady-state velocities are smaller than predicted from simple, Michaelis-Menten kinetics. (3) The first-order rate constant for the approach to steady-state has a complex dependence on substrate concentration and decreases sigmoidally as substrate concentration increases. These results indicate that the 20S proteasome is a hysteretic enzyme and is subject to substrate inhibition. To explain these observations we propose a minimal kinetic model with two critical mechanistic features: (1) the 20S proteasome has two cooperative active sites for Suc-Leu-Leu-Val-Tyr-AMC and (2) there are two interconvertible conformers of active 20S proteasome. To probe this mechanism in greater detail, we explored the kinetic mechanism of inhibition of the 20S proteasome-catalyzed hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC by the peptide aldehyde, Ac-Leu-Leu-Nle-H. Our studies reveal a nonlinear dependence of reciprocal steady-state velocity on inhibitor concentration (i.e., parabolic inhibition) as well as a nonlinear dependence of the apparent inhibitor dissociation constant on substrate concentration. Both of these observations are explained by binding of inhibitor at multiple sites on the enzyme. Taken together, the results of this study indicate that the 20S proteasome is a conformationally flexible protein that can adjust to the binding of ligands and that has multiple and cooperative active sites. These results support a view of the proteasome's substrate specificity in which (1) substrates are recognized and hydrolyzed by more than one active site; (2) each active site can bind substrates that possess a variety of P1 residues; and (3) the P1 residue plays a relatively minor role as a specificity determinant. Finally, we interpret the results of this study to suggest that, in vivo, the 20S proteasome requires conformational plasticity for its interactions with regulatory complexes and, after it has combined with appropriate regulatory complexes, to catalyze hydrolysis of proteins.
Assuntos
Quimotripsina/metabolismo , Cisteína Endopeptidases/metabolismo , Modelos Teóricos , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/enzimologia , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/isolamento & purificação , Cinética , Matemática , Dados de Sequência Molecular , Complexos Multienzimáticos/isolamento & purificação , Oligopeptídeos , Complexo de Endopeptidases do Proteassoma , Coelhos , Dodecilsulfato de Sódio/farmacologia , Especificidade por SubstratoRESUMO
Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Lactonas/farmacologia , Complexos Multienzimáticos/metabolismo , Reticulócitos/enzimologia , Acetilcisteína/química , Acetilcisteína/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Cisteína Endopeptidases/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Lactonas/química , Estrutura Molecular , Complexos Multienzimáticos/isolamento & purificação , Complexo de Endopeptidases do Proteassoma , Coelhos , StreptomycesRESUMO
We have investigated the specificity of isopeptidase T toward peptide-AMC substrates based on the C-termini of ubiquitin. The substrates investigated were Z-Gly-Gly-AMC, Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC and were hydrolyzed by isopeptidase T with kc/Km values of < 0.1, 1, 18, and 95 M-1 s-1, respectively. In the course of these experiments, we observed that the hydrolytic activity of isopeptidase T toward these substrates is modulated by ubiquitin in a biphasic fashion. While submicromolar concentrations of ubiquitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of kc/Km varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC, respectively. Kd for ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 microM. At higher concentrations, ubiquitin is inhibitory and titrates kc/Km with an average Ki value of 3.0 +/- 1.3 microM for all three substrates. To explain these observations, we propose a structural model for isopeptidase T that involves two binding sites for ubiquitin. We propose that the two sites are adjacent to one another and are the extended active site that binds two ubiquitin moieties of a polyubiquitin chain for isopeptide bond hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Carbono-Nitrogênio Liases , Liases/metabolismo , Ubiquitinas/farmacologia , Sequência de Aminoácidos , Catálise , Endopeptidases/metabolismo , Hidrólise , Cinética , Liases/efeitos dos fármacos , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
We have investigated the inhibition of the human matrix metalloproteinase stromelysin (SLN) by the N-(carboxyalkyl)dipeptide Ala[N]hPhe-Leu-anilide and find that it is a competitive, slow-binding inhibitor of this enzyme with Ki = 3 x 10(-8) M (pH 6.0, 25 degrees C). The dependence of k(obs), the observed first-order rate constant for the approach to steady state, on Ala[N]hPhe-Leu-anilide concentrations less than 10(-5) M is linear and suggests a simple, one-step mechanism with kon = 3.4 x 10(4) M-1 s-1 and k(off) = 1.2 x 10(-3) s-1 (pH 6.0, 25 degrees C). Using rapid kinetic techniques, we extended the concentration range of Ala[N]hPhe-Leu-anilide to 2 x 10(-3) M and found that the [Ala[N]hPhe-Leu-anilide] dependence of K(obs) suggests saturation kinetics with a Ki' near 5 x 10(-4) M. Detailed analysis of these data reveal that the dependence of k(obs) on [Ala[N]hPhe-Leu-anilide] is, in fact, sigmoidal. To probe the chemical mechanism of inhibition, we determined pH and temperature dependencies and solvent deuterium isotope effects. For k(on), delta H not equal to = 12.4 kcal/mol and -T delta S not equal to = 6.2 kcal/mol (T = 298 K; [I]steady-state = 10(-6) M), while for k(off), delta H not equal to = 12.5 kcal/mol and -T delta S not equal to = 8.9 kcal/mol (T = 298 K). pH dependencies of the kinetic parameters for inhibition are complex but reflect greater potency at lower pH and suggest a mechanism involving the same active-site groups that are involved in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dipeptídeos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Sequência de Aminoácidos , Ligação Competitiva , Deutério , Dipeptídeos/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Metaloproteinase 3 da Matriz , Dados de Sequência Molecular , Temperatura , TermodinâmicaRESUMO
Substrate specificity studies from this laboratory suggested that Ac-Pro-Leu-Ala-Nva-TrpNH2, and its thioester derivative, Ac-Pro-Leu-Ala-SNva-TrpNH2, would be substrates for stromelysin (SLN). In this paper, we report that both peptides are efficiently hydrolyzed not only by SLN but also by two other matrix metalloproteinases, collagenase and gelatinase, and by the bacterial metalloproteinase thermolysin. The pH-dependence of kc/Km for the SLN-catalyzed hydrolysis of Ac-Pro-Leu-Ala-SNva-TrpNH2 is identical to pH-dependencies for peptide hydrolysis and suggests no major mechanistic differences between thioester and amide hydrolysis by SLN.
Assuntos
Metaloendopeptidases/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Clostridium/enzimologia , Colagenases/metabolismo , Fibroblastos/enzimologia , Gelatinases/metabolismo , Humanos , Hidrólise , Cinética , Matemática , Metaloproteinase 3 da Matriz , Modelos Teóricos , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Compostos de Sulfidrila/metabolismo , Termolisina/metabolismoRESUMO
We have investigated the inhibition of the human matrix metalloproteinase stromelysin (SLN) by the peptide phosphonamidate, phthaloyl-N-(CH2)4-P(O2-)-Ile-(beta-naphthyl)Ala-NH-CH3, and find that it is a potent, slow-binding inhibitor of SLN with kon = 2.7 x 10(4) M-1 sec-1, koff = 1.9 x 10(-4) sec-1, and Ki = 7 nM (pH 5.0, 25 degrees C). To probe the mechanism of inhibition, we determined pH-dependencies and solvent deuterium isotope effects. pH-dependencies of the kinetic parameters for inhibition are complex but reflect greater inhibitory potency at lower pH and suggest a mechanism for inhibition that involves the same active site groups as are involved in catalysis. The solvent isotope on kon (kon, H2O/Kon,D2O) is normal and equals 1.5 +/- 0.1. Together with the pH-dependence of inhibition, this value suggests that kon is rate-limited by a process that involves general-acid/general-base catalysis. We propose that kon is rate-limited by general-acid catalyzed ligand exchange of inhibitor for the zinc-bound water molecule.
Assuntos
Dipeptídeos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Compostos Organofosforados/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Deutério , Humanos , Concentração de Íons de Hidrogênio , Cinética , Metaloproteinase 3 da Matriz , Dados de Sequência Molecular , Ensaio Radioligante , Proteínas RecombinantesRESUMO
To probe the specificity of the metalloendoproteinase stromelysin toward peptide substrates, we determined kc/Km values for the stromelysin-catalyzed hydrolyses of peptides whose design was based loosely on the structure of a known SLN substrate, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2, hydrolysis at Gln-Phe, kc/Km = 1700 M-1 s-1). Several noteworthy points emerge from this study: (i) Catalytic efficiency is dependent on peptide chain length with N-terminal truncation of substance P resulting in more pronounced rate-constant reductions than C-terminal truncation. These results suggest the existence of an extended active site for stromelysin. (ii) Preferences at positions P3, P2, P1, P1', and P2' are for the hydrophobic amino acids Pro, Leu, Ala, Nva, and Trp, respectively. (iii) Investigation of specificity at P3' supports our earlier hypothesis that SLN has a requirement for a hydrogen-bond donor at this position in its substrates. Based on these observations, we designed and had synthesized the fluorogenic substrate N-(2,4-dinitrophenyl)Arg-Pro-Lys-Pro-Leu-Ala-Nva-TrpNH2, whose stromelysin-catalyzed hydrolysis can be monitored continuously (kc/Km = 45,000 M-1 s-1).
Assuntos
Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Catálise , Fluorometria , Humanos , Cinética , Metaloproteinase 3 da Matriz , Metaloendopeptidases/química , Dados de Sequência Molecular , Peptídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
To probe the mechanism of stromelysin (SLN)-catalyzed peptide hydrolysis, we determined the pH dependence of kc/Km and solvent deuterium isotope effects on kc and kc/Km. pH dependencies of kc/Km were determined for the SLN-catalyzed hydrolysis of three peptides: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2,Arg-Pro-Ala-Pro-Gln-Gln- Phe-Phe - Gly-Leu-NleNH2, and N-acetyl-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2 (cleavage at Gln-Phe bond). The pH dependencies are all bell-shaped with shoulders that extend from pH 7.5 to 8.5. The existence of a shoulder indicates that the reaction mechanism involves at least two routes to products. These curves are governed by three proton ionizations with pKa values of 5.4, 6.1, and 9.5. The solvent isotope effect measurements provided the following values: D(kc/Km) = 0.80 +/- 0.05 and D(kc) = 1.58 +/- 0.05. That D(kc/Km) and D(kc) are different suggests that the rate-limiting transition states for the processes governed by kc/Km and kc cannot be the same. We use these results, together with analogy to thermolysin catalysis, to develop a mechanism for SLN catalysis.
Assuntos
Metaloendopeptidases/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Catálise , Deutério , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Metaloproteinase 3 da Matriz , Dados de Sequência Molecular , Oligopeptídeos/química , SolventesRESUMO
The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.