Clusterin is a specific stabilizer and liberator of extracellular cathepsin K

The cysteine peptidase cathepsin K is a major player in extracellular proteolysis. Here we describe the identification of the multifunctional extracellular chaperone clusterin as a cathepsin K-binding protein. Clusterin increases the stability of cathepsin K in dilute solution and in the presence of high protein concentration. It does not alter the activity of the enzyme but acts as a liberator by preventing substrate inhibition. Kinetic measurements show that clusterin binds cathepsin K with high affinity (K(d) = 0.5-0.6 nM). Altogether these results provide novel insights into the mechanisms involved in the fine-tuning of cysteine cathepsin activity in the extracellular space.     

Substrate recognition by the human fatty-acid synthase

The human fatty-acid synthase (HFAS) is a potential target for anti-tumor drug discovery. As a prelude to the design of compounds that target the enoyl reductase (ER) component of HFAS, the recognition of NADPH and exogenous substrates by the ER active site has been investigated. Previous studies demonstrate that modification of Lys-1699 by pyridoxal 5'-phosphate results in a specific decrease in ER activity. For the overall HFAS reaction, the K1699A and K1699Q mutations reduced kcat and kcat/KNADPH by 8- and 600-fold, respectively (where KNADPH indicates the Km value for NADPH). Thus, Lys-1699 contributes 4 kcal/mol to stabilization of the rate-limiting transition state following NADPH binding, while also stabilizing the most stable ground state after NADPH binding by 3 kcal/mol. A similar effect of the mutations on the ER partial reaction was observed, in agreement with the proposal that Lys-1699 is located in the ER NADPH-binding site. Most unexpectedly, however, both kcat and kcat/KNADPH for the beta-ketoacyl reductase (BKR) reaction were also impacted by the Lys-1699 mutations, raising the possibility that the ER and BKR activities share a single active site. However, based on previous data indicating that the two reductase activities utilize distinct cofactor binding sites, mutagenesis of Lys-1699 is hypothesized to modulate BKR activity via allosteric effects between the ER and BKR NADPH sites.     

Allosteric Hotspots in the Main Protease of SARS-CoV-2

Inhibiting the main protease of SARS-CoV-2 is of great interest in tackling the COVID-19 pandemic caused by the virus. Most efforts have been centred on inhibiting the binding site of the enzyme. However, considering allosteric sites, distant from the active or orthosteric site, broadens the search space for drug candidates and confers the advantages of allosteric drug targeting. Here, we report the allosteric communication pathways in the main protease dimer by using two novel fully atomistic graph-theoretical methods: Bond-to-bond propensity, which has been previously successful in identifying allosteric sites in extensive benchmark data sets without a priori knowledge, and Markov transient analysis, which has previously aided in finding novel drug targets in catalytic protein families. Using statistical bootstrapping, we score the highest ranking sites against random sites at similar distances, and we identify four statistically significant putative allosteric sites as good candidates for alternative drug targeting.     

Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid.

The cysteine protease cathepsin K is believed to play a key role in bone resorption as it has collagenolytic activity and is expressed predominantly and in high levels in bone resorbing osteoclast cells. The addition of nitric oxide (NO) and NO donors to osteoclasts in vitro results in a reduction of bone resorption, although the mechanism of this effect is not fully understood. The S-nitroso derivatives of glutathione (GSNO) and N-acetylpenicillamine (SNAP) and the non-thiol NO donors NOR-1 and NOR-3 all inhibited the activity of purified cathepsin K in a time- and concentration-dependent manner (IC(50) values after 15 min of preincubation at pH 7.5 of 28, 105, 0.4, and 10 microM, respectively). Cathepsin K activity in Chinese hamster ovary cells stably transfected with cathepsin K was also inhibited by the above NO donors with similar potencies. GSNO at 100 microM also completely inhibited the autocatalytic maturation at pH 4.0 of procathepsin K to cathepsin K. The inhibition of cathepsin K by GSNO was rapidly reversed by DTT, but inhibition by NOR-1 was not reversed by DTT, and analysis of the inhibited cathepsin K for S-nitrosylation using the Greiss reaction gave negative results in both cases. Analysis of the protein by electrospray liquid chromatography/mass spectrometry showed that the inhibition of cathepsin K by GSNO resulted in a mass increase of 306 +/- 2 Da, consistent with the formation of a glutathione adduct. Prior inhibition of cathepsin K by the active site thiol-modifying inhibitor E-64 blocked the modification by GSNO, indicating that the glutathione adduct is likely formed at the active site cysteine. Treatment of cathepsin K with NOR-1 resulted in a mass increase of between 30 and 50 Da, corresponding to the oxidation of a cysteine to sulfinic and sulfonic acids. Cotreatment of cathepsin K with NOR-1 plus the sulfenic acid reagent dimedone resulted in a mass increase of approximately 141 Da, which is consistent with the formation of a dimedone adduct. This result demonstrates that the NOR-1-dependent formation of cathepsin K sulfinic and sulfonic acids occurs via a sulfenic acid. These results show that inhibition of cathepsin K activity and its autocatalytic maturation represent two potential mechanisms by which NO can exert its inhibitory effect on bone resorption. This work also shows that oxidative thiol modifications besides S-nitrosylation should be considered when the effects of NO and NO donors on critical thiol-containing proteins are investigated.     

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K(m), but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25°C but concealed at 37°C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25°C, but not at 37°C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.     

Modulation of hypotensive effects of kinins by cathepsin K

Kinins are pro-inflammatory peptides, which participate in the maintenance of cardiovascular homeostasis, and play a key role in numerous diseases, including lung fibrosis and hypertension. Evidence has been provided recently for the presence of alternative mechanisms of bradykinin generation and/or degradation. Here we showed that cathepsin K may act as a potent kinin-degrading enzyme in bloodstream. Contrary to cathepsin L, cathepsin K attenuates kallikrein-induced decrease of rat blood pressure, and reduces the hypotensive effect of bradykinin in a dose-dependent manner. Moreover, we identified, by engineering the S2 subsite of both recombinant enzymes, two critical residues involved respectively in the kininase activity of cathepsin K, i.e. Tyr67/Leu205, versus kininogenase activity of cathepsin L, i.e. Leu67/Ala205. In conclusion, according to its ability to modulate hypotensive effects of kinins, we propose that cathepsin K is a kininase of biological relevance, in complement of well-documented neutral endopeptidase or angiotensin-converting enzyme.     

Arylamine based cathepsin K inhibitors: investigating P3 heterocyclic substituents

A modification of novel cathepsin K inhibitors I was carried out. The structural design was aimed at reducing the lipophilic character of compounds I for obtaining better pharmacokinetic profiles. This modification afforded several less lipophilic compounds with good inhibitory activities and pharmacokinetic profiles, although the enzyme selectivity over cathepsin S was left at issue.     

Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5-p-fluorosulfonylbenzoyl adenosine.

The purine nucleotide derivative, 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSO₂B_ZAdo) functions as an affinity label for the allosteric sites of phosphofructokinase. The modified enzyme at pH 6.9 is insensitive to allosteric inhibition by ATP, activation by AMP, c-AMP, ADP and shows no sigmoidal kinetics for fructose-6-P. The reaction does not appear to occur at the catalytic site since modification of the enzyme does not significantly affect its specific activity nor its Michaelis constant at pH 8.2. ADP, and to a much lesser degree AMP and ATP, protects the enzyme from modification by the adenosine reagent. The modified enzyme essentially does not bind significant amounts of AMP, c-AMP, ADP, but still binds an analog of ATP, AppNHp. The adenosine affinity label will be of value in studies on the nature of the AMP-ADP allosteric sites.     

Selective inhibition of the collagenase activity of cathepsin K

Cathepsin K, the main bone degrading protease, and chondroitin 4-sulfate (C4-S) form a complex with enhanced collagenase activity. In this report, we demonstrate the specific inhibition of the collagenase activity of cathepsin K by negatively charged polymers without affecting the overall proteolytic activity of the protease. Three different mechanisms to interfere with cathepsin-catalyzed collagen degradation are discussed: 1) inhibition of the formation of the cathepsin K/C4-S complex, 2) inhibition of the attachment of C4-S to collagen, and 3) masking of the collagenase cleavage sites in collagen. By targeting these interaction sites, collagen degradation can be modulated while the non-collagenolytic activities of cathepsin K remain intact. The main inhibitory effect on collagen degradation is due to the impeding effect on the active cathepsin K/C4-S complex. Essential structural elements in the inhibitor molecules are negative charges which compete with the sulfate groups of C4-S in the cathepsin K/C4-S complex. The inhibitory effect can be controlled by length and charge of the polymers. Longer negatively charged polymers (e.g. polyglutamates, oligonucleotides) tend to inhibit all three mechanisms, whereas shorter ones preferentially affect the cathepsin K/C4-S complex.     

Modification of the conformational equilibria in the sodium and potassium dependent adenosinetriphosphatase with glutaraldehyde

Glutaraldehyde treatment of electroplax membrane preparations of Na,K-ATPase leads to irreversible changes in the enzymic behavior of the protein, which are not due to modification of the active site. When the glutaraldehyde treatment is carried out in a medium containing K+ and without Na+, the "K+-modified enzyme" so produced shows the following changes in enzymic properties: The steady-state phosphorylation by ATP and the rate of ATP-ADP exchange are decreased to approximately 40% of control, while Na,K-ATPase activity decreases to approximately 15% of control. Phosphatase activity is decreased very little, but the potassium activation parameters of the reaction are changed, from K0.5 approximately equal to 5 mM and nH = 1.9 in control to K0.5 approximately equal to 0.5 mM and nH = 1 in K+-modified enzyme. KI(app) for nucleotide inhibition of phosphatase activity is increased significantly. Changes in the cation dependence of the ATPase reaction are also observed. All of these effects can be explained by assuming that the cross-linking of surface groups in protein subunits when they are in conformation E2 shifts the intrinsic conformational equilibrium of the enzyme toward E2. We considered the simplest mathematical model for the coupling between K+ binding and the conformational equilibrium, with equivalent potassium sites that must be simultaneously in the same state. If one assumes that the potassium activation of phosphatase activity in the K+-modified enzyme reflects the affinity for K+ of E2, the behavior of the phosphatase activity in the native enzyme can be fit if there are only two potassium sites, whose affinity is 80-fold higher in E2 than in E1, and the equilibrium constant for E2 in equilibrium E1 is about 250. The same sites can explain the activation of dephosphorylation during ATP hydrolysis. Independent of the model chosen, potassium ions must be required for the catalytic action of form E2 and cannot be merely "allosteric activators". The enzyme modified with glutaraldehyde in a medium containing Na+ also has interesting properties, but their rationalization is less straightforward. The Na,K-ATPase activity is inhibited more than the "partial reactions", as in the K+-modified enzyme. We suggest that this is a generally expected result of modifications of the enzyme.     

Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K

The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+. The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E. coli K with equal efficiency. In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor. Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation. The rate of enzyme activation was determined using the AdoMet analog Sinefungin. The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions.     

Identification of potential allosteric binding sites in cathepsin K based on intramolecular communication

Network theory methods and molecular dynamics (MD) simulations are accepted tools to study allosteric regulation. Indeed, dynamic networks built upon correlation analysis of MD trajectories provide detailed information about communication paths between distant sites. In this context, we aimed to understand whether the efficiency of intramolecular communication could be used to predict the allosteric potential of a given site. To this end, we performed MD simulations and network theory analyses in cathepsin K (catK), whose allosteric sites are well defined. To obtain a quantitative measure of the efficiency of communication, we designed a new protocol that enables the comparison between properties related to ensembles of communication paths obtained from different sites. Further, we applied our strategy to evaluate the allosteric potential of different catK cavities not yet considered for drug design. Our predictions of the allosteric potential based on intramolecular communication correlate well with previous catK experimental and theoretical data. We also discuss the possibility of applying our approach to other proteins from the same family.     

Protection by Alcuronium of Muscarinic Receptors Against Chemical Inactivation and Location of the Allosteric Binding Site for Alcuronium

Abstract: We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [³H]methyl- N -scopolamine ([³H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [³H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues ( p -nitrobenzenesulfonyl fluoride and 4-chloro-7-nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N' -dicyclohexylcarbodiimide, and N -ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [³H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [³H]NMS binding sites against the inactivation by tyrosine- and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site.     

Determinants of allosteric activation of yeast pyruvate kinase and identification of novel effectors using computational screening

We have analyzed the structural determinants of the allosteric activation of yeast pyruvate kinase (YPK) by mutational and kinetic analysis and initiated a structure-based design project to identify novel effectors that modulate its allosteric response by binding to the allosteric site for fructose-1,6-bisphosphate (FBP). The wild-type enzyme is strongly activated by fructose-1,6-bisphosphate and weakly activated by both fructose-1-phosphate and fructose-6-phosphate; the strength of the activation response is proportional to the affinity of the allosteric effector. A point mutation within the 6'-phosphate binding loop of the allosteric site (T403E) abolishes activation of the enzyme by fructose-1, 6-bisphosphate. The mutant enzyme is also not activated by F1P or F6P. The mutation alone (which incorporates a glutamic acid that is strictly conserved in mammalian M1 isozymes) slightly reduces cooperativity of substrate binding. Three novel compounds were identified that effect the allosteric regulation of YPK by FBP and/or act as novel allosteric activators of the enzyme. One is a physiologically important diphospho sugar, while the other two are hydrophobic compounds that are dissimilar to the natural effector. These results demonstrate that novel allosteric effectors may be identified using structure-based screening and are indicative of the potential of this strategy for drug discovery. Regulatory sites are generally more divergent than catalytic sites and therefore offer excellent opportunities for discrimination and specificity between different organisms or between different tissue types.     

Allosteric effects of some antifibrinolytic amino acids on the catalytic activity of human plasmin.

The effects of L-lysine, 6-aminohexanoic acid, and trans-4-aminomethylcy-clohexane-1-carboxylic acid on the catalytic activity of plasmin (EC 3.4.21.7) have been investigated. The kinetics of the plasmin-catlysed hydrolysis of alpha-N-benzoyl-L-arginine ethyl ester in the presence of these compounds have been studied at a number of different concentrations of the three modifiers. They each exert two effects on the reaction, an activation and an inhibition, the concentration dependencies of which are markedly different. They must therefore arise from two different interactions between plasmin and the modifier. The inhibition is competitive, so that it most probably results from direct interaction at the catalytic site. The activation is kinetically non-competitive. The experimental observations seem to be explained best by assuming that L-lysine and certain analogous compounds function as both allosteric modifiers and competitive inhibitors of plasmin.     

Species differences between human and rat in the substrate specificity of cathepsin K

Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.     

13C and 15N isotope effects as a probe of the chemical mechanism of Escherichia coli aspartate transcarbamylase.

13C and 15N isotope effects have been measured for the aspartate transcarbamylase (ATCase) reaction in an effort to elucidate the chemical mechanism of this highly regulated enzyme. The observed 15(V/K(asp))H2O value for the ATCase holoenzyme at saturing carbamyl phosphate and 12 mM L-aspartate is 1.0045 at pH 7.5, and this value remains unchanged in the presence of 5 mM ATP (activator) or 5 mM CTP (inhibitor). The fact that the isotope effect is not changed by the allosteric modifiers supports the conclusion that the kinetic properties of the active form of ATCase are not influenced by ATP or CTP. The observed 15(V/K(asp)) values for the catalytic subunit of ATCase are also the same as those determined for the holoenzyme, suggesting that the chemical mechanisms of both enzyme species are the same. Quantitative analysis of 13C and 15N isotope effects in both H2O and D2O has led to the proposal of a chemical model for the ATCase reaction which involves a precatalytic conformational change to form an activated complex that facilitates deprotonation of L-aspartate by an enzyme functional group. Nucleophilic attack on the carbonyl carbon of carbamyl phosphate by the alpha-amino group of L-aspartate results in the formation of a tetrahedral intermediate. An intramolecular proton transfer leads to formation of products N-carbamyl-L-aspartate and inorganic phosphate.     

Cathepsin M: a lysosomal proteinase with aldolase-inactivating activity

A proteinase, designated cathepsin M, that catalyzes the limited modification and inactivation of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from rabbit liver. On the basis of its molecular size (M_r = 30,000), activation by sulfhydryl compounds and inhibition by leupeptin it has been characterized as a B-type cathepsin, but other properties distinguish it from cathepsins B, L, and H. Approximately 50% of the total cathepsin M activity is associated with membranes prepared from disrupted lysosomes; this fraction of the activity is also expressed by intact lysosomes. In the membrane-bound form the enzyme is active at neutral pH, but the soluble enzyme and the activity eluted from the membranes are maximally active at pH 5.0. Fasting increases the activity of cathepsin M; the increase is almost entirely in the membrane-bound fraction.     

Linked-function origins of cooperativity in a symmetrical dimer

The thermodynamic origins of substrate binding cooperativity in a dimeric enzyme that can bind one substrate (A) and one allosteric ligand (X) to each of two identical subunits are discussed. It is assumed that maximal activity is not subject to allosteric modification and that the substrates and allosteric ligands achieve binding equilibrium in the steady state. Each uniquely ligated form is assumed to be capable of exhibiting unique binding properties, and only the principles of thermodynamic linkage are used to constrain the system further. The explicit relationship between the Hill coefficient, the concentration of X, and the magnitudes of the relevant coupling free energies and dissociation constants is derived. In the absence of X only the homotropic coupling between substrate sites contributes to a nonhyperbolic substrate saturation profile. An allosteric ligand, X, can alter the cooperativity in two distinct ways, one mechanism being manifested when X is saturating and the only only when X is present at saturating concentrations. By evaluating the concentration of substrate required to produce half-maximal velocity as a function of [X], as well as the Hill coefficients when X is absent and fully saturating, the dissociation and coupling constants most important for understanding the mechanisms of allosteric action in an enzyme of this type can be determined.     

The topology of the glutamine and ATP binding sites of human asparagine synthetase.

Human asparagine synthetase was examined using a combination of chemical modifiers and specific monoclonal antibodies. The studies were designed to determine the topological relation between the nucleotide binding site and the glutamine binding site of the human asparagine synthetase. The purified recombinant enzyme was chemically modified at the glutamine binding site by 6-diazo-5-oxo-L-norleucine (DON), and at the ATP binding site by 8-azidoadenosine 5'-triphosphate (8-N3ATP). The effects of chemical modification with DON included a loss of glutamine-dependent reactions, but no effect on ATP binding as measured during ammonia-dependent asparagine synthesis. Similarly, modification with 8-N3ATP resulted in a loss of ammonia-dependent asparagine synthesis, but no effect on the glutaminase activity. A series of monoclonal antibodies was also examined in relation to their epitopes and the sites modified by the two covalent chemical modifiers. It was found that several antibodies were prevented from binding by specific chemical modification, and that the antibodies could be classified into groups correlating to their relative binding domains. These results are discussed in terms of relative positions of the glutamine and ATP binding sites on asparagine synthetase.