Azapeptides as inhibitors and active site titrants for cysteine proteinases

Ester and amide derivatives of alpha-azaglycine (carbazic acid, H2NNHCOOH), alpha-azaalanine, and alpha-azaphenylalanine (i.e., Ac-l-Phe-NHN(R)CO-X, where X = H, CH3, or CH2Ph, respectively) were synthesized and evaluated as inhibitors of the cysteine proteinases papain and cathepsin B. The ester derivatives inactivated papain and cathepsin B at rates which increased dramatically with leaving group hydrophobicity and electronegativity. For example, with 8 (R = H, X = OPh) the apparent second-order rate constant for papain inactivation was 67 600 M-1 s-1. Amide and P1-thioamide derivatives do not inactivate papain, nor are they substrates; instead they are weak competitive inhibitors (0.2 mM < Ki < 4 mM). Inactivation of papain involves carbamoylation of the enzyme, as demonstrated by electrospray mass spectrometry. Active site titration indicated a 1:1 stoichiometry for the inactivation of papain with 8, and both inactivated papain and cathepsin B are highly resistant to reactivation by dialysis (t1/2 > 24 h at 4 degrees C). Azaalanine derivatives Ac-L-Phe-NHN(CH3)CO-X inactivate papain ca. 400- 900-fold more slowly than their azaglycine analogues, consistent with the planar configuration at Nalpha of the P1 residue and the very substantial stereoselectivity of papain for L- vs D- residues at the P1 position of its substrates. Azaglycine derivative 9 (R = H, X = OC6H4NO2-p) inactivates papain extremely rapidly (>70 000 M-1 s-1), but it also decomposes rapidly in buffer with release of nitrophenol (kobs = 0.13 min-1); under the same conditions 8 shows <7% hydrolysis over 24 h. This nitrophenol release probably involves cyclization to an oxadiazolone since 17 (R = CH3, X = OC6H4NO2-p), which cannot form an isocyanate, releases nitrophenol almost as rapidly (kobs = 0.028 min-1). Cathepsin C, another cysteine proteinase with a rather different substrate specificity (i.e., aminopeptidase), was not inactivated by 8, indicating that the inactivation of papain and cathepsin B by azapeptide esters is a specific process. Their ease of synthesis coupled with good solution stability suggests that azapeptide esters may be useful as active site titrants of cysteine proteinases and probes of their biological function in vivo.     

The role of the active site cysteine in catalysis by type 1 iodothyronine deiodinase

Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mM concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in K(i)propylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mM reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.     

Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity

Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).     

Fluorescent labeling of cysteine 39 on Escherichia coli primase places the dye near an active site

Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine. Its high chemical reactivity is likely due to its proximity to primase's zinc, which is probably ligated to the adjacent residues 40-62. The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive. Primase is rapidly, site-specifically modified by fluorescein maleimide (FM) at this cysteine. Modification with FM at this residue does not lead to any activity loss in a coupled RNA/DNA synthesis assay, indicating that it is not a catalytically essential residue. In contrast, iodoacetamidefluorescein (IAF) modifies cysteine 39 more slowly and stoichiometrically inhibits activity. It was not clear why these two similar fluorescent dyes should have such different inhibitory effects when attached to the same cysteine. The IAF inhibition must be due to some property of the link between the fluorescein and the cysteine because that is how it differs from FM. The pKa's of the fluoresceins from both FM- and IAF-modified primase are strongly shifted to lower values (approximately 5.4) compared to free fluorescein. These results strongly suggest that the deprotonated form of the fluoresceins are stabilized on primase by a strong interaction with the adjacent zinc in the zinc finger motif. The ability to place a noninhibitory FM at this site will be of great assistance in fluorescence energy transfer studies since the distances established to cysteine 39 will reflect the distance to the essential zinc finger motif.     

Mutation of cis-proline 207 in mitochondrial creatine kinase to alanine leads to increased acid stability

SETTING: Hamad General Hospital, the tertiary health centre for Doha, Qatar. OBJECTIVE: The purpose of the study was to define and correlate the role of radiology with clinical and pathological findings in abdominal tuberculosis. MATERIAL AND METHODS: A total of 59 patients (47 males and 12 females) diagnosed bacteriologically and/ or histologically for abdominal tuberculosis were radiologically assessed. Evaluation was based on the analysis of plain abdominal radiographs, gastro-intestinal contrast studies (barium meal follow through and barium enema), ultrasonography and computed tomography. RESULTS: Plain abdominal radiographs performed in 38 patients were positive in 19 cases (50%). Gastrointestinal contrast examinations were positive in 27 out of 34 cases (80%). Ultrasound examinations were abnormal in 25 out of 31 cases (81%), while computed tomography, performed in 24 patients, revealed abnormal findings in 19 cases (80%). Combined radiographic and imaging procedures revealed peritoneal involvement (ascites) in 16 patients (27%), bowel involvement in 36 (61%), mass lesion in 11 (19%), lymphadenopathy in 13 (22%) and organ involvement in 13 (22%). CONCLUSION: There was no single radiological method that provided all necessary information suggestive of abdominal tuberculosis. Although unequivocal diagnosis of abdominal tuberculosis can only be made by culture and histological findings, combined computed tomography and ultrasound findings were the most important imaging tools in the diagnostic process for abdominal tuberculosis, while contrast studies helped to assess the extent of bowel disease, hence influencing decisions concerning surgery.     

Mutation of an active site residue of tryptophan synthase (beta-serine 377) alters cofactor chemistry

To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta-Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa approximately 7.7) from a form with a protonated internal aldimine nitrogen (lambdamax = 416 nm) to a deprotonated form (lambdamax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta2 subunit and alpha2 beta2 complex are pH-independent. The reaction of the S377D alpha2 beta2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambdamax = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.     

Three-dimensional structures of the cysteine proteases cathepsins K and S deduced by knowledge-based modelling and active site characteristics

Human cathepsins K and S are recently identified proteins with high primary sequence homology to members of papain superfamily, including cathepsins B, L, H and papain. Models of the tertiary structures of cathepsins K and S and their complexes with a specific substrate and inhibitor were constructed and compared with the recently determined X-ray structure of cathepsin K. A major problem in the determination of the three-dimensional structure of proteins concerns the quality of the structural models obtained from the interpretation of experimental data. The framework of the tertiary structures of cathepsins K and S consisted of structurally conserved regions from the tertiary structure of the papain superfamily and the variable regions were constructed with fragments of other proteins from the protein data base. Based on docking studies the non-bonded interaction energies of ligands with the cathepsins were estimated. These energies correlate with experimentally determined substrate and inhibitory potency.     

Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo

DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.     

Pharmacological approaches to counter the toxicity of Dopa

The biocompatibility of a 3 mm band made of polyethylene terephthalate (Trevira hochfest) has been tested in an experimental study within right knee joints of 60 sheep. After transsecting the anterior cruciate ligament (ACL), two randomized groups were formed. In group I, the ACL was repaired according to the Marshall technique whilst in group II an additional 3 mm polyethylene terephthalate (PET) augmentation band was implanted using the through-the-condyle (TTC) procedure. To assess the biocompatibility of the augmentation device the knee joints of both groups were punctured and the synovial fluids were analyzed before, as well as 2, 6, 16, 26, and 52 weeks after the operation. In addition, the histologic appearance of excised suprapatellar pouches and ipsilateral inguinal and popliteal lymphatic noduli were examined. Comparing both groups no significant differences were found neither before nor after the augmented and non-augmented ACL repair. No pathological increase in the total protein concentration occurred after operation and no significant differences versus the preoperative analysis were found. No synovitis signalling a decrease in the glucose concentration was observed. The cytological examination revealed no increase of the leukocyte cell count results. Within the synovial specimen neither free nor phagocytosed PET wearparticles could be detected. In groups I and II the histological appearance of excised popliteal and inguinal lymphatic noduli showed a normal result. In 25% of the PET augmented ACL repairs, a slight concentration of PET wearparticles and solitary, multinuclear giant foreign body cells could be seen in the histological preparations of suprapatellar pouches.     

Mutation of a major keratin phosphorylation site predisposes to hepatotoxic injury in transgenic mice

Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament (IF) proteins. One important physiologic function of K8/18 is to protect hepatocytes from drug-induced liver injury. Although the mechanism of this protection is unknown, marked K8/18 hyperphosphorylation occurs in association with a variety of cell stresses and during mitosis. This increase in keratin phosphorylation involves multiple sites including human K18 serine-(ser)52, which is a major K18 phosphorylation site. We studied the significance of keratin hyperphosphorylation and focused on K18 ser52 by generating transgenic mice that overexpress a human genomic K18 ser52--> ala mutant (S52A) and compared them with mice that overexpress, at similar levels, wild-type (WT) human K18. Abrogation of K18 ser52 phosphorylation did not affect filament organization after partial hepatectomy nor the ability of mouse livers to regenerate. However, exposure of S52A-expressing mice to the hepatotoxins, griseofulvin or microcystin, which are associated with K18 ser52 and other keratin phosphorylation changes, resulted in more dramatic hepatotoxicity as compared with WT K18-expressing mice. Our results demonstrate that K18 ser52 phosphorylation plays a physiologic role in protecting hepatocytes from stress-induced liver injury. Since hepatotoxins are associated with increased keratin phosphorylation at multiple sites, it is likely that unique sites aside from K18 ser52, and phosphorylation sites on other IF proteins, also participate in protection from cell stress.     

The five cysteine residues located in the active site region of bovine aspartyl (asparaginyl) beta-hydroxylase are not essential for catalysis

In previous chemical modification studies on bovine aspartyl (asparaginyl) beta-hydroxylase, cysteines were implicated as critical catalytic residues. Using site-directed mutagenesis, the five cysteine residues located in a highly conserved region of the enzyme identified as the active site were individually mutated to alanine. Substitutions at cysteine 637, 644, 656, 681, and 696 resulted in active mutant enzymes indicating that these residues are not required for catalysis.     

Identification of the oxidation states of the active site cysteine in a recombinant protein tyrosine phosphatase by electrospray mass spectrometry using on-line desalting

The oxidation state of the cysteine residue at the active site of human protein tyrosine phosphatase (PTP-1B) greatly affects its enzymatic activity. We wished to examine peroxide-treated preparations for modifications of this enzyme with electrospray mass spectrometry in order to determine the locations and oxidation states of the cysteines or other residues involved in the process. Since these reaction products contained large amounts of salts and buffers, they required desalting prior to analysis. Existing on- and off-line methods presented certain difficulties in handling and sample usage. Based on recent experience with direct syringe admission of sample, we developed a procedure as a simple, inexpensive alternative to full high-performance liquid chromatography systems that provides on-line desalting using only a few microL of sample. The method was applied to the analysis of oxidized PTP-1B preparations where conversion of cysteine 215 to both sulfinic and sulfonic acid residues was demonstrated.     

Kinetic mechanism of active site non-equivalence in transketolase

The two-step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non-equivalence in TDP binding.     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant kon, for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction.     

Chronic ethanol treatment leads to increased ornithine decarboxylase activity: implications for a role of polyamines in ethanol dependence and withdrawal

STUDY OBJECTIVES: To determine intrasubject and intersubject variability in, and the effects of food and antacids on, the pharmacokinetics of pyrazinamide (PZA). DESIGN: Randomized, four-period, crossover phase I study. SUBJECTS: Fourteen healthy men and women volunteers. INTERVENTIONS: Subjects ingested single doses of PZA 30 mg/kg under fasting conditions twice, without a high-fat meal and with an aluminum-magnesium antacid. They also received standard dosages of isoniazid, rifampin, and ethambutol. MEASUREMENTS AND MAIN RESULTS: Serum was collected for 48 hours and assayed by gas chromatography with mass selective detector. Data were analyzed by noncompartmental methods and a compartmental analysis using nonparametric expectation maximization. Both fasting conditions produced similar results: mean PZA Cmax 53.4+/-10.4 microg/ml, Tmax 1.43+/-1.06 hours, and AUC(0-infinity) 673+/-79.7 microg x hr/ml. Fasting results are similar to those in previous reports. In the presence of antacids, subjects had a mean Cmax of 55.6+/-9.0 microg/ml, Tmax of 1.43+/-1.23 hours, and AUC(0-infinity) of 628+/-88.4 microg x hr/ml. In the presence of the high-fat meal, mean Cmax was 45.6+/-9.44 pg/ml, Tmax 3.09+/-1.74 hours, and AUC(0-infinity) 687+/-116 microg x hr/ml. CONCLUSIONS: These small changes in Cmax, Tmax, and AUC(0-infinity) can be avoided by giving PZA on an empty stomach whenever possible.     

Unique fold and active site in cytomegalovirus protease

Human herpesviruses are responsible for a variety of diseases. They are divided into three subfamilies: alpha includes herpes simplex viruses (HSV-1 and HSV-2) and varicella-zoster virus (VZV); beta includes cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6); and gamma includes Epstein-Barr virus (EBV). Each virus encodes a serine protease that is essential for its replication and is a potential target for therapeutic intervention. Human CMV is a ubiquitous opportunistic pathogen that can result in life-threatening infections in congenitally infected infants, immunocompromised individuals and immunosuppressed cancer or transplant patients. Here we report the crystal structure of human CMV protease at 2.5 angstroms resolution. The structure reveals a fold that has not been reported for any other serine protease, and an active site consisting of a novel catalytic triad in which the third member is a histidine instead of an aspartic acid, or possibly a catalytic tetrad consisting of a serine, two histidines and an aspartic acid. An unusual dimer interface that is important to the protease activity has also been identified.     

Protein components contribute to active site architecture for eukaryotic ribonuclease P

In eukaryotes, ribonuclease P (RNase P) requires both RNA and protein components for catalytic activity. The eukaryotic RNase P RNA, unlike its bacterial counterparts, does not possess intrinsic catalytic activity in the absence of holoenzyme protein components. We have used a sensitive photoreactive cross-linking assay to explore the substrate-binding environment for different eukaryotic RNase P holoenzymes. Protein components from the Tetrahymena thermophila and human RNase P holoenzymes form specific products in photoreactions containing [4-thio]-uridine-labeled pre-tRNAGln. The HeLa RNase P RNA in neither the presence nor the absence of holoenzyme protein components formed cross-link products to the pre-tRNAGln probe. Parallel photo-cross-linking experiments with the Escherichia coli RNase P holoenzyme revealed that only the bacterial RNase P RNA forms specific substrate photoadducts. A protein-rich active site for the eukaryotic RNase P represents one unique feature that distinguishes holoenzyme organization between bacteria and eukaryotes.     

Homologies in the active site regions of lactate dehydrogenases

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.     

Contribution to activity of histidine-aromatic, amide-aromatic, and aromatic-aromatic interactions in the extended catalytic site of cysteine proteinases

Within the papain family of cysteine proteinases few other residues in addition to the catalytic triad, Cys25-His159-Asn175 (papain numbering) are completely conserved [Berti & Storer (1995) J. Mol. Biol. 246, 273-283]. One such residue is tryptophan 177 which participates in a Trp-His-type interaction with the catalytic His159. In all enzymes of this class for which a three-dimensional structure has been reported, an additional highly conserved tryptophan, Trp181, also interacts with Trp177 via an aromatic-aromatic interaction in which the planes of the indole rings are essentially perpendicular. Also, both indole rings participate as pseudo-hydrogen bond acceptors in interactions with the two side chain amide protons of Asn175. Clearly, the proximity of Trp177 and Trp181 to the catalytic triad residues His159 and Asn175 and their network of interactions points to potential contributions of these aromatic residues to catalysis. In this paper, using cathepsin S, a naturally occurring variant that has a phenylalanine residue at position 181, we report the kinetic characterization of mutants of residues 175, 177, and 181. The results are interpreted in terms of the side chain contributions to catalytic activity and thiolate-imidazolium ion-pair stability. For example, the side chain of Asn175 has a major influence on the ion-pair stability presumably through its hydrogen bond to His159. The magnitude of this effect is modulated by Trp177, which shields the His159-Asn175 hydrogen bond from solvent. The His159-Trp177 interaction also contributes significantly to ion-pair stability; however, Trp181 and its interactions with Asn175 and Trp177 do not influence ion-pair stability to a significant degree. The observation that certain mutations at positions 177 and 181 result in a reduction of kcat/Km but do not appear to influence ion-pair stability probably reflects the contributions of these residues to substrate binding.