Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis–Menten mechanism (original) (raw)
Related papers
Cysteine protease attribute of eukaryotic ribosomal protein S4
Biochimica et Biophysica Acta (BBA) - General Subjects, 2012
Background: Ribosomal proteins often carry out extraribosomal functions. The protein S4 from the smaller subunit of Escherichia coli, for instance, regulates self synthesis and acts as a transcription factor. In humans, S4 might be involved in Turner syndrome. Recent studies also associate many ribosomal proteins with malignancy, and cell death and survival. The list of extraribosomal functions of ribosomal proteins thus continues to grow. Methods: Enzymatic action of recombinant wheat S4 on fluorogenic peptide substrates Ac-XEXD↓-AFC (Nacetyl-residue-Glu-residue-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR↓-AMC (N-CBZ-Phe-Argaminomethylcoumarin) as well as proteins has been examined under a variety of solution conditions. Results: Eukaryotic ribosomal protein S4 is an endoprotease exhibiting all characteristics of cysteine proteases. The K m value for the cleavage of Z-FR↓-AMC by a cysteine mutant (C41F) is about 70-fold higher relative to that for the wild-type protein under identical conditions, implying that S4 is indeed a cysteine protease. Interestingly, activity responses of the S4 protein and caspases toward environmental parameters, including pH, temperature, ionic strength, and Mg 2 + and Zn 2 + concentrations, are quite similar. Respective kinetic constants for their cleavage action on Ac-LEHD↓-AFC are also similar. However, S4 cannot be a caspase, because unlike the latter it also hydrolyzes the cathepsin substrate Z-FR↓-AMC. General significance: The eukaryotic S4 is a generic cysteine protease capable of hydrolyzing a broad spectrum of synthetic substrates and proteins. The enzyme attribute of eukaryotic ribosomal protein S4 is a new phenomenon. Its possible involvement in cell growth and proliferations are presented in the light of known extraribosomal roles of ribosomal proteins.
Biochemical Journal, 2000
We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for oaminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S % and S $ subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S # h and S $ h. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P % , P $ , P # h and P $ h were made. The S % to S # h subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S $ h, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S % , S $ , S # h and S $ h of the three enzymes.
FEBS Letters, 1988
High hydrophobicity of the second amino acid N-terminal to the scissile bond (P, residue) is generally considered to be the major factor in the specificity of the substrates for cysteine proteases of the papain family. To examine the catalytic contribution of the S,P, hydrogen bond apparent from X-ray crystallographic studies, the kinetics of Z-Phe-Gly-OEt and its thiono derivative were compared. The thiono compound contains a sulfur atom in place of the carbonyl oxygen of the phenylalanine residue. It was found that the specificity rate constants for the reactions of the thiono substrate with various cysteine proteases are lower by 2-3 orders of magnitude as compared to the corresponding rate constants for the 0x0 substrate. This remarkable effect is not expected in the light of previous studies indicating that the change from oxygen to sulfur in the P, residue was without an appreciable effect. The results are interpreted in terms of a distorted binding of the thiono substrate.
Unusual Role of a Cysteine Residue in Substrate Binding and Activity of Human AP-Endonuclease 1
Journal of Molecular Biology, 2008
The mammalian AP-endonuclease (APE1) repairs abasic (AP) sites and strand breaks with 3' blocks in the genome that are formed both endogenously and as intermediates during base excision repair (BER). APE1 has an unrelated activity as a redox activator (and named Ref-1) for several trans-acting factors. In order to identify whether any of the seven cysteine residues in human APE1 (hAPE1) affects its enzymatic function, we substituted these singly or multipully with serine. The repair activity is not affected in any of the mutants except those with C99S mutation. The Ser99-containing mutants lost affinity for DNA and its activity was inhibited by 10 mM Mg 2+ . However, the Ser99 mutant has normal activity in 2 mM Mg 2+ . Using crystallographic data and molecular dynamic (MD) simulation, we have provided a mechanistic basis for the altered properties of the C99S mutant. We earlier predicted that Mg 2+ with potential binding sites A and B, bound at the B-site of WT APE1substrate complex and moves to the A-site after cleavage occurs, as observed in the crystal structure. The APE1-substrate complex is stabilized by a H-bond between His309 and the AP-site in the C99S mutant. We now show that this bond is broken to destabilize the complex in the absence of the Mg 2+ . This effect due to the mutation of Cys99, ~16Å from the active site, on the DNA binding and activity is surprising.
A Conserved Arginine Plays a Role in the Catalytic Cycle of the Protein Disulphide Isomerases
Journal of Molecular Biology, 2004
The pK a values of the CXXC active-site cysteine residues play a critical role in determining the physiological function of the thioredoxin superfamily. To act as an efficient thiol-disulphide oxidant the thiolate state of the N-terminal cysteine must be stabilised and the thiolate state of the C-terminal cysteine residue destabilised. While increasing the pK a value of the C-terminal cysteine residue promotes oxidation of substrates, it has an inhibitory effect on the reoxidation of the enzyme, which is promoted by the formation of a thiolate at this position. Since reoxidation is essential to complete the catalytic cycle, the differential requirement for a high and a low pK a value for the C-terminal cysteine residue for different steps in the reaction presents us with a paradox. Here, we report the identification of a conserved arginine residue, located in the loop between b5 and a4 of the catalytic domains of the human protein disulphide isomerase (PDI) family, which is critical for the catalytic function of PDI, ERp57, ERp72 and P5, specifically for reoxidation. An examination of the published NMR structure for the a domain of PDI combined with molecular dynamic studies suggest that the side-chain of this arginine residue moves into and out of the active-site locale and that this has a very marked effect on the pK a value of the active-site cysteine residues. This intra-domain motion resolves the apparent dichotomy of the pK a requirements for the C-terminal active-site cysteine.
Journal of Combinatorial Chemistry, 1999
To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S 3 subsite and hydrophobic residues in S 2 ; the most prevalent residue not being Phe but Val. The S 1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S 1 ′-S 4 ′ subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the k cat /K M 's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO 2 )PMPPLCTSMK(Abz) (k cat /K M ) 2109 (mM s) -1 ), Y(NO 2 )PYAVQSPQK(Abz) (k cat /K M ) 1524 (mM s) -1 ), and Y(NO 2 )PVLRQQRSK-(Abz) (k cat /K M ) 1450 (mM s) -1 ). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft. † 1X, set as standards100 times dilution of enzyme (8.34 µM); 20X, 20 times dilution of 1X enzyme solution; Abz, 2-
ACS Omega
Cruzain, a cysteine protease of the papain family, is essential in the development of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease, making it an attractive target for developing new drugs. The present paper is aimed at the study of the catalytic mechanism of the cruzain by first exploring the different protonation states of the active site Cys25 and His159 in the Michaelis complex and the effect on the full catalytic mechanism of this enzyme. The exploration of the equilibrium between these two states has been performed with alchemical free energy perturbation methods with molecular mechanics (MM) force fields and by generating the free energy surfaces in terms of the potential of mean force computed at two levels of theory: AM1d/MM and M06-2X/6-31+G(d,p):AM1d/MM. Alternative mechanisms for the acylation step have been identified on the free energy surfaces and the results suggest the existence of three new reaction mechanisms starting from the peptide binding to the apoenzyme in its neutral Cys25S/His159 dyad state. The mechanism starting with the protonation of the nitrogen atom of the peptide followed by the attack of Cys25S − was revealed as the most favorable one, but it can be competitive with its counterpart mechanism initiated in the Cys25S − /His159H + ion pair Michaelis complex. Analysis of energetic and average geometries will allow continuing improvement of our knowledge on this enzyme at the molecular level, which can be crucial to the design of new inhibitors based on the structures of the transition states (transition states analogues) or stable intermediates.
PLOS Computational Biology, 2015
Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function.
Structural Biology of Cysteine Biosynthetic Pathway Enzymes
Amebiasis, 2014
The cysteine biosynthetic pathway is of central importance for the growth, survival, and pathogenicity of the anaerobic protozoan parasite Entamoeba histolytica . This pathway is present across all species but is absent in mammals. Cysteine, the product of this pathway, is the only antioxidative thiol responsible for fi ghting oxidative stress in E. histolytica . Serine acetyl transferase (SAT) and O -acetyl serine sulfhydrylase (OASS) are the two enzymes catalyzing the de novo cysteine biosynthetic pathway. In all organisms in which so far this pathway is known to exist, both these enzymes associate to form a regulatory complex, but in E. histolytica this complex is not formed. The cysteine biosynthetic pathway has been optimized in this organism to adapt to and fulfi ll its cysteine requirements.
Design of a new selective cysteine protease inactivator and its mechanistic implications
Bioorganic & Medicinal Chemistry Letters, 1995
Cbz-Phe-epoxide was designed as a selective inactivator of cysteine proteases. It exhibits a time-and concentration-dependent inactivation of cysteine proteases, while showing no activity towards serine proteases. The inhibition is irreversible, correlated with loss of the five active-site thiol, and its rate is at least 104 faster than the rate of a model reaction in solution. These results support the proposed active-site directed, protonationdependent, mechanism-based mode of inactivation of cysteine proteases by the new inhibitor. Proteolytic enzymes can be divided into four families, based on their active site residues and their mechanism of catalysis. 1 Two of these families, namely the serine-and cysteine proteases share many characteristics; They both utilize a covalent nucleophilic catalysis, and involve formation of an acyl cnzyme and two tetrahedral intermediates along the catalytic pathway. It was recently suggested that x~ bile in scrine proteases the nucleophilic attack of the serine alkoxide precedes the protonation of the leaving amino group (thus, leading to a negatively charged tetrahedral intermediate), the corresponding attack of the cysteine thiolate in cysteine p,'oteases is subsequent to or concomitant with protonation of the substrate (leading to a ncutral tetrahedral intermediate). 2 This may explain the selectivity of peptidyl diazomethanes as inhibitors of cysteine proteases. 3'4 This subtle distinction (if found to be correct) between serine-and cysteine proteases may serve as the basis for the design of selective inhibitors for cysteine protcases. Furthermore. if a compound designed to challange the proposed mechanistic difference exhibits such selectivity towards cysteine proteases-it would support the suggested modification in cysteine protease catalytic mechanism. While epoxides are stable compounds, 5 they become highly electrophilic upon protonation. This principle was used recently in the development of an inhibitor for carboxypeptidase A, a metallo protease in which Zn +2 can activate the epoxidic moiety. 6 It is probably also a dominant factor in making E-64 and its derivatives efficient general inbibitors of most cysteine proteases, 7 though it was recently suggested that protonation of the epoxide in this case is carried out by a water molecule rather than by the active site histidine, g Weak inhibition of the cysteine protease cathepsin B was observed, upon its incubation with epoxides derived from allyl amine. 9 Other epoxides were also demonstrated to inactivate a variety of enzymes, in which activation of the epoxide is mechanistically feasible. 1° N-protected-or-amino epoxides (Figure 1), derived from the corresponding natural or-amino acids, are good analogs of the corresponding substrates of either serine or cysteine proteases and therefore are expected to bind "normally" in their respective binding sites. However, if the suggested mechanistic difference between serine-and cysteine proteases (initial nucleophilic attack in the former vs. initial protonation in the latter) is