Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors.

The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.

Purification and characterization of cysteine protease from Pleurotus ostreatus.

Cysteine protease activity in mycelial culture increased 7.7-fold after fruit body formation in Pleurotus ostreatus, using the Leu pNA (LPNA) cleavage assay. The enzyme was purified from fruit bodies and its M(r) was 97,000 by gel filtration and 48,500 by SDS-PAGE, indicating that it is a dimer. The enzyme was sensitive to iodoacetic acid, p-chloromercuribenzoate, N-ethylmaleimide, and HgCl2. The sequence of the first 9 N-terminal amino acids of cysteine protease was ASGLXXAIL.

Extracellular dextran-induced p-nitrophenyl-alpha-D-glucoside-hydrolyzing enzyme of Bacillus circulans KA-304: a producer of Schizophyllum commune-lytic enzyme.

p-NP-alpha-D-Glucoside-hydrolyzing activity in the culture filtrate of Bacillus circulans KA-304, a producer of Schizophyllum commune cell-wall lytic enzyme, increased remarkably when the bacterium was grown on dextran as a carbon source. It was suggested that the increase of the activity was caused by increases of two major species, alpha-D-glucosidase I and alpha-D-glucosidase II. alpha-D-Glucosidase I, which showed a certain reactivity toward dextran, was isolated from the filtrate (MW 70 kDa, 35-fold, 10% recovery). The enzyme was stable around pH 6.5-7.5 and showed its highest activity at pH 6.5. The enzyme preparation inactivated with p-chloromerucuribenzoic acid recovered its activity by incubating with ditiothereitol. Its substrate specificity suggested that the enzyme was an exo-type enzyme with certain affinity toward alpha-1,6-glucosidic linkage.

Bovine kidney low molecular weight acid phosphatase: FMN-dependent kinetics.

A low molecular weight bovine kidney acid phosphatase, electrophoretically homogeneous and with a relative molecular mass of 17.8 kDa, was used in this work. Among the various substrates tested, FMN was found to be the most effective, at pH 7.0. Distinct activation energy values were obtained for p-nitrophenyl phosphate- (45.44 kJ mol-1) and flavin mononucleotide- (28.60 kJ mol-1) hydrolysis reactions. The FMN hydrolysis was strongly inhibited by Cu2 and pCMB, but activated by guanosine. Pyridoxal-phosphate and vanadate were competitive inhibitors for the FMN-dependent reaction.

Identification of residues in the translocation pathway of EmrE, a multidrug antiporter from Escherichia coli.

EmrE is a small, 12-kDa, highly polyspecific antiporter, which exchanges hydrogen ions with aromatic cations such as methyl viologen. EmrE-mediated transport is inhibited by the sulfhydryl-reactive reagent 4-(chloromercuri)benzoic acid (PCMB) but not by a variety of other sulfhydryl reagents. This differential effect is due to the fact that the organic mercurial is a substrate of the transporter and can reach domains otherwise inaccessible to the different reagents. To find out which of the three cysteine residues in EmrE is reacting with PCMB, each was replaced with serine and it was shown that none of them is essential for transport activity. A protein completely devoid of Cys residues (CL) is also capable of substrate accumulation albeit at a slower rate. Mutated proteins in which only one of the native cysteines was left whereas the other changed to serine were also constructed. The use of these proteins demonstrated that two of the three Cys in EmrE, Cys-41 and Cys-95, but not Cys-39, react with PCMB. A related mercurial, 4-(chloromercuri)benzenesulfonic acid (PCMBS), is only a very poor inhibitor, probably because of the negative charge it bears. PCMBS reacts with EmrE in an asymmetric and unique way. It reacts with the mutant bearing a single Cys residue in position 95 (CL-C95) only when the reagent is present in the outside face of the membrane and with the mutant CL-C41 only when allowed to permeate to the cell interior; as expected, it does not react with the mutant protein bearing a single Cys at position 39 (CL-C39). It is concluded that PCMB permeates through the substrate pathway of EmrE and covalently reacts with the two exposed residues, Cys-95 and Cys-41, but not with Cys-39, located on the opposite face of the helix relative to residue 41. In addition, because of the asymmetric reactivity to PCMBS, an inhibitor that does not permeate through the protein, it is concluded that Cys-41 is closer to the cytoplasmic face than Cys-95. The results demonstrate the existence of a domain accessible only to substrates and provide a unique tool for studying the substrate permeation pathway of an ion-coupled transporter.

X-ray diffraction of a cysteine-containing bacteriorhodopsin mutant and its mercury derivative. Localization of an amino acid residue in the loop of an integral membrane protein.

We have used heavy-atom labeling and X-ray diffraction to localize a single amino acid in the integral membrane protein bacteriorhodopsin (bR). To provide a labeling site, we used the bR mutant, A103C, which contains a unique cysteine residue in the short loop between transmembrane alpha-helices C and D. The mutant protein was expressed in and purified from Halobacterium halobium, where it forms a two-dimensional crystalline lattice. In the lattice form, the protein reacted with the sulfhydryl-specific reagent p-chloromercuribenzoate (p-CMB) in a 1:0.9 stoichiometry to yield the p-mercuribenzoate derivative (A103C-MB). The functional properties of A103C and A103C-MB, including the visible absorption spectrum, light-dark adaptation, photocycle, and proton release kinetics, were similar to those of wild-type bR. X-ray diffraction experiments demonstrated that A103C and A103C-MB membranes have the same hexagonal protein lattice as wild-type purple membrane. Thus, neither the cysteine substitution nor mercury labeling detectably affected bR structure or function. By using Fourier difference methods, the in-plane position of the mercuribenzoate label was calculated from intensity differences in the X-ray diffraction patterns of A103C and A103C-MB. This analysis revealed a well-defined mercury peak located between alpha-helices C and D. The approach reported here offers promise for refining the bR structural model, for monitoring conformational changes in bR photointermediates, and for studying the structure of other proteins in two-dimensional crystals.

[Interaction of spin labels and probes with Na+,K+-ATPase]. / Issledovanie vzaimodeistviia spinovykh metok i zondov s Na+,K+-ATPazoi.

Localization of the PCMB-R spin label and benzocarboline probe bound with the purified preparation of pig kidney-Na+, K(+)-ATPase relative to active site of the enzyme was studied by EPR method. The number of Mn2+ ions in active site of the enzyme as well as that bound with lipids was determined from EPR spectra of paramagnetic manganese ions replacing magnesium ions were measured in frozen protein samples of Na2+, K(+)-ATPase at 77 K. It has been found that sulfhydryl group of the enzyme modified by PCMB-R and benzocarboline probe are placed at distances 38 A and 50 A, respectively, from Mn2+ ions in the active site of Na+, K(+)-ATPase. Evaluation of the immersion depth of the nitroxyl radical into protein globule showed that benzocarboline probe was immobilized near the macromolecular protein surface; there are two bound probe sites, distinguished by accessibility of ferricyanide ions.

[Enzymes of a bacteriolytic lysoamidase preparation. Various properties of bacteriolytic protease L2]. / Fermenty bakterioliticheskogo preparata lizoamidaza. Nekotorye svoistva bakterioliticheskoi proteinazy L2.

Bacteriolytic proteinase L2 is able to cleave fluorogenic synthetic tripeptide anthranoyl-alanyl-alanyl-phenylalanyl-nitroanilide (Abz-Ala-Ala-Phe-pNA) at the bond between phenylalanine and p-nitroaniline. Optimal conditions of the tripeptide cleavage have been determined: pH 6.7 + 0.1; mu = 2 (by NaCl); t = 40 degrees C; KM = 2.6 x 10(-5) M. Metal cations reduced the enzyme activity. The enzyme was inhibited by EDTA, p-CMB, DIF. The synthetic tripeptide can be used to determine the activity of the L2 enzyme.

A convenient synthesis of N-succinimidyl-3-iodo-[125I]benzoate, a reagent for protein iodination.

A convenient procedure has been developed for the synthesis of N-succinimidyl-3-iodo-[125I]benzoate. The procedure involved the synthesis of chloromercuribenzoic acid, its esterification with N-hydroxysuccinimide (NHS) and exchanging the mercury moiety with radioactive iodine in the presence of an oxidant. The obtained product was attached to human serum albumin and its stability was compared with Chloramine-T (Ch-T) radioiodinated protein. The results indicated that the reagent-radiolabeled protein was stable for longer periods and the deiodination rate was significantly lower.

Regulation of aspartate carbamoyltransferase in Neisseria and Branhamella species.

The regulatory characteristics of aspartate carbamoyltransferase (ACTase EC 2.1.3.2) from various species of Neisseria and Branhamella have been compared. Great differences in the regulatory nature of the enzymes were observed. ATP and GTP were positive effectors in Neisseria meningitidis, N. gonorrhoeae and nine other coccal "true neisseriae" species. In four "false neisseriae" species, including Branhamella catarrhalis, no stimulating effect of ATP or GTP was observed. The rod-shaped N. elongata behaved as the "false neisseriae" in these respects, despite its taxonomic affinity to the coccal "true neisseriae" species. Except in N. meningitidis and N. lactamica, CTP had no distinct stimulatory effect. CTP had a strong inhibitory effect on ACTases from N. elongata and the "false neisseriae" species N. caviae and B. catarrhalis. The inhibitory effect of CTP was weak in N. cinerea, N. denitrificans, and the "false neisseriae" species N. ovis and N. cuniculi. Thus, there was no sharp reflection of taxonomy in the regulation of ACTase by CTP in these groups of bacteria. The apparent [S]0.5 values for aspartate and carbamoyl phosphate, displayed for five of the eighteen species, showed great variability with [S]0.5 values for aspartate ranging from 6 to 34, and for carbamoyl phosphate from 2 to 9. Treatment of the enzyme from the main test microbe N. meningitidis strain M1 by heat or para-chloromercuribenzoate (pCMB) showed that both the catalytic and the regulatory functions decreased in parallel as in the class A enzymes found in species of Pseudomonas. An estimation of the molecular weight (Mr) of the ACTase enzyme from N. meningitidis showed it to be about 295,000, which resembles the class B enzymes found in the Enterobacteriaceae.