Role reversal for substrates and inhibitors. Slow inactivation of D-amino acid transaminase by its normal substrates and protection by inhibitors

D-Amino acid transaminase, which catalyzes the synthesis of D-alanine and D-glutamate for the bacterial cell wall, is a candidate for the design of specific inhibitors that could be novel antimicrobial agents. Under the experimental conditions usually employed for enzyme assays, kinetic parameters for its substrates were determined for short incubation periods, when intermediates and products do not accumulate and the enzyme activity is linear with time. Such kinetic analyses indicate that the enzyme accepts most D-amino acids but D-aspartate and D-glutamate are the best substrates. Under a different type of experimental conditions when the enzyme is exposed to D-alanine, intermediates, and products for periods of hours, it slowly becomes inactivated (Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., and Manning, J. M. (1992) Biochemistry 31, 6018-6023). We now report that D-aspartate, D-glutamate, and L-alanine also lead to slow inactivation. Methylation or amidation of the alpha-COOH group of D-alanine prevents inactivation, indicating that decarboxylation is required for inactivation; the slow release of CO2 from substrate is demonstrated. The alpha-methyl analog of D-alanine, D-aspartate, and D-glutamate do not lead to inactivation, showing that the alpha-hydrogen of the substrate is required, i.e. that some processing is required. Lys145, which binds pyridoxal 5'-phosphate in the wild-type enzyme, is not involved in the inactivation since two active site mutant enzymes, K145Q and K145N, are also inactivated. Reactivation of the inactive enzyme at acidic pH is accompanied by the release of ammonia corresponding to 1 mol/mol of dimeric enzyme. Competitive inhibitors, amine-containing buffers, and thiols effectively impede the inactivation. This reversal in the roles of substrates and inhibitors, i.e. when a substrate can be an inactivator and an inhibitor can act as a protector, occurs during a time period not usually used to measure steady-state kinetics or initial velocities of enzyme reactions and could have physiological relevance in cells.     

Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase

The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 angstroms from the coenzyme, pyridoxal 5'-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP --> E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.     

Characterization of the genes encoding D-amino acid transaminase and glutamate racemase, two D-glutamate biosynthetic enzymes of Bacillus sphaericus ATCC 10208

In Bacillus sphaericus and other Bacillus spp., D-amino acid transaminase has been considered solely responsible for biosynthesis of D-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding D-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28. 8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the D-amino acid transaminase of the thermophilic Bacillus sp. strain YM1.     

Effects of the E177K mutation in D-amino acid transaminase. Studies on an essential coenzyme anchoring group that contributes to stereochemical fidelity

D-Amino acid transaminase is a bacterial enzyme that uses pyridoxal phosphate (PLP) as a cofactor to catalyze the conversion of D-amino acids into their corresponding alpha-keto acids. This enzyme has already been established as a target for novel antibacterial agents through suicide inactivation by a number of compounds. To improve their potency and specificity, the detailed enzyme mechanism, especially the role of its PLP cofactor, is under investigation. Many PLP-dependent transaminases have a negatively charged amino acid residue forming a salt-bridge with the pyridine nitrogen of its cofactor that promotes its protonation to stabilize the formation of a ketimine intermediate, which is subsequently hydrolyzed in the normal transaminase reaction pathway. However, alanine racemase has a positively charged arginine held rigidly in place by an extensive hydrogen bond network that may destabilize the ketimine intermediate, and make it too short-lived for a transaminase type of hydrolysis to occur. To test this hypothesis, we changed Glu-177 into a titratable, positively charged lysine (E177K). The crystal structure of this mutant shows that the positive charge of the newly introduced lysine side chain points away from the nitrogen of the cofactor, which may be due to electrostatic repulsions not being overcome by a hydrogen bond network such as found in alanine racemase. This mutation makes the active site more accessible, as exemplified by both biochemical and crystallographic data: CD measurements indicated a change in the microenvironment of the protein, some SH groups become more easily titratable, and at pH 9.0 the PMP peak appeared around 315 nm rather than at 330 nm. The ability of this mutant to convert L-alanine into D-alanine increased about 10-fold compared to wild-type and to about the same extent as found with other active site mutants. On the other hand, the specific activity of the E177K mutant decreased more than 1000-fold compared to wild-type. Furthermore, titration with L-alanine resulted in the appearance of an enzyme-substrate quinonoid intermediate absorbing around 500 nm, which is not observed with usual substrates or with the wild-type enzyme in the presence of L-alanine. The results overall indicate the importance of charged amino acid side chains relative to the coenzyme to maintain high catalytic efficiency.     

Probing immunogenicity of a T cell epitope by L-alanine and D-amino acid scanning

All residues of the I-Ed restricted fragment 24-36 of a snake toxin were individually changed into L-alanine and the corresponding D-enantiomer. Four analogs substituted with L-Ala at positions 25;30, 31 and 33, and nine analogs substituted with a D-residue along the stretch 25-33 lost most (position 28) or all their capacity to stimulate a toxin-specific T hybridoma. None of these analogs stimulated splenocytes from mice immunized with the peptide 24-36. Only the L-A31 and D-W29 modified analogs could prime a T cell response which, however, showed no cross-reactivity with the native peptide, demonstrating that T cell response selectivity can be deeply modified by mutation or configuration inversion of a single residue. Our data suggest that (i) the region 25-33 is the core of the T epitope that binds to I-Ed, and (ii) Y25 R30 and R33 contribute to the peptide binding by anchoring into pockets of I-Ed. In agreement with T cell priming observations, only the L-A31 and D-W29 modified analogs elicited strong antibody responses, just like the peptide 24-36, whereas nearly all other analogs were less immunogenic. All but the L-Ala30 and L-Ala33 modified analogs were recognized by a 24-36 specific antiserum as well as the native peptide. Altogether, our results show that substitution by D-amino acid in a peptide could be particularly well-suited for either minimizing the risk of hypersensitivity or designing peptidic vaccines.     

Structure of D-amino acid oxidase: new insights from an old enzyme

D-amino acid oxidase is the prototype of flavin-dependent oxidases. The recent resolution of its 3D structure has provided an explanation for several of its properties and has led to a substantial revision of the mechanism of D-amino acid dehydrogenation, with significant implications for the general understanding of flavin-dependent catalysis.     

Phytanic acid alpha-hydroxylation by bacterial cytochrome P450

The science of drug metabolism, like any other science, has advanced from simple beginnings (by today's standards) to its present state. One can examine the path that has been taken to understand the forces driving the direction of evolution of this science. The trends discovered can then be used to make reasonable extrapolations about the changes that might be expected in the future. That exercise is the subject of this article. The main focus will be on drug metabolism as practiced in the industrial environment, representing the author's main experience as well as the principal arena of practical applications of the science. The discussion will draw mainly on broad phenomena occurring in this application of drug metabolism to drug discovery and development.     

Inactivation of the phosphoenolpyruvate-dependent phosphotransferase system in various species of bacteria by vinylglycolic acid

The alkenoic hydroxyacid 2-hydroxy-3-butenoic acid (vinylglycolate) specifically inhibited the phosphotransferase system in a variety of bacteria while not affecting respiration-coupled transport systems.     

Inactivation of alpha-ketoglutarate dehydrogenase during oxidative decarboxylation of alpha-ketoadipic acid

alpha-Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of alpha-ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of alpha-ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.     

Inactivation of rifampin by Nocardia brasiliensis

Rifampin was glycosylated by a pathogenic species of Nocardia, i.e., Nocardia brasiliensis. The structures of two glycosylated compounds (RIP-1 and RIP-2) isolated from the culture broth of the bacterium were determined to be 3-formyl-23-(O-[beta-D-glucopyranosyl])rifamycin SV and 23-(O-[beta-D-glucopyranosyl])rifampin, respectively. Both compounds lacked antimicrobial activity against other gram-positive bacteria as well as the Nocardia species.     

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by ferrylmyoglobin

This article describes the development and psychometric evaluation of self-report measurement instruments in (nursing) research. The aim of this study is to gain more insight into, and understanding of the use of such instruments. To be more specific, this paper deals with: (1) What is a self-report measurement instrument?; (2) How to develop such an instrument?; (3) What is its psychometric quality in terms of validity and reliability?; (4) How to analyze an instrument statistically?; (5) Which are the pros en cons of a self-report measurement instrument in general?; and (6) Where do we find examples of good measurement instruments? These six questions will be answered with the help of several practical research examples. The article concludes with a few suggestions for literature concerning existing measurement instruments and their psychometric qualities.     

Inactivation of coliphage Q beta by potassium ferrate

The study looked at palpitations in relation to the prevalence of arrhythmia, as assessed by 24-h ambulatory electrocardiography (ECG) in a population sample. The subjects were randomly drawn from among those involved in a cardiovascular survey. Forty-three of those who answered 'Yes' and 54 of those who answered 'No' (84% of those eligible) to the following question, participated: 'Have you observed sudden changes in your heart rate or heart rhythm during the preceding year?' In both groups mean age was 49 years and 58% were men. There was no relationship between recorded arrhythmia and perceived palpitations during monitoring. The prevalence of at least one arrhythmic episode (ventricular or supraventricular arrhythmia or pauses > = 1.5 s) was significantly higher in those who had perceived palpitations during the previous year (98%) than in those who had not (74%) (P < 0.0014). Through a simple question about palpitations during the preceding year we were able to identify significantly a population with true arrhythmias. However, the question could not be used to define a population entirely without arrhythmia. The high prevalence of arrhythmia in subjects without reported palpitations indicates that it is a normal finding which alone should not demand further clinical investigations.     

MS2 Inactivation by Chloride-Assisted Electrochemical Disinfection

An electrochemical (EC) disinfection system employing an iridium–antimony–tin-coated titanium anode and direct current was used to inactivate bacteriophage MS2 in synthetic solutions with sodium chloride addition. The inactivation data fit the modified Chick–Watson (n ≠ 1) model well. The model indicates that, although better disinfection could be achieved with increases in salt content, contact time, and applied current, these three parameters influence the EC disinfection of MS2 in distinct manners and to different degrees. Compared with chlorination, our EC disinfection system exhibited superior inactivation capability especially with a longer contact time or in the presence of ammonium. The formation of trihalomethanes and haloacetic acids in the EC system was smaller than that from chlorination but a large formation of chlorate ions was observed. These differences indicate that the EC system is likely to produce other potent oxidants that enhance inactivation and alter disinfection by-product formation.     

Inactivation of hamster monomorphic N-acetyltransferase by vinyl fluorenyl ketone

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that play important roles in the detoxification and activation of xenobiotic arylamines and their metabolites. Vinyl fluorenyl ketone (VFK) is a selective and potent active site-directed irreversible inhibitor of rat liver monomorphic NAT. The present study demonstrated that VFK is an active site-directed affinity label for hamster liver monomorphic NAT, but is a much less effective inactivator of the polymorphic N-acetyltransferase isozyme. The potency, irreversibility and selectivity of VFK make it a potentially valuable tool for characterization of NATs that exhibit acetyl donor specificity similar to that of hamster monomorphic NAT.     

Inactivation of respiratory syncytial virus by detergents and disinfectants

The activity of a number of detergents and disinfectants against respiratory syncytial virus (RSV) was evaluated in an in vitro assay system. Equal volumes of RSV and serial 10-fold dilutions of the test agents were mixed at 4 degrees C for 5 minutes. The RSV titer in each mixture was compared with that of untreated RSV alone. In 14 experiments with input RSV titers ranging from 2.6 x 10(3) to 2 x 10(7) plaque-forming units/ml, a 10-fold dilution of 5.25% sodium hypochlorite (generic bleach) inactivated (> or = 3-log reduction in titer) the virus. With lower RSV titers inactivation was also observed at a 100-fold dilution of bleach. Fetal calf serum concentrations up to 50% as an organic load did not diminish the bleach effect. The degree of RSV inactivation was also defined for Lysol, povidone-iodine, Amphyl, Hibiclens, Osyl, ethanol and Listermint. The short contact time, the reproducible nature of the findings and the continued effectiveness with increasing organic loads all suggest that detergents and disinfectants can potentially play an important role in decreasing the spread of RSV infection.     

Inactivation of Bacillus Spores by Ultraviolet or Gamma Radiation

Bacillus anthracis spores represent an important bioterrorism agent that can be dispersed in air or water. Existing decontamination practices based on these spores have focused on chemical disinfectants; however, the basic characteristics of radiation-based disinfectants suggest potential advantages in their application for control of Bacillus spores. Experiments were conducted to examine the effectiveness of ultraviolet (UV254) radiation and γ radiation for inactivation of Bacillus spores. Spores of Bacillus cereus were used for most experiments because of their similarity to B. anthracis. A limited number of experiments were also conducted using B. anthracis Sterne spores. In aqueous suspension, B. anthracis Sterne spores were observed to be slightly more resistant to UV254 than the spores of B. cereus. For the conditions of culture and assay used in these experiments, both spore types were more sensitive to UV254 radiation in aqueous suspension than the spores of B. subtilis, which are commonly used to characterize the performance of UV disinfection systems for water. Dried spores on surfaces were observed to be more resistant to UV254 than the same spores in aqueous suspension; it is likely that the increased resistance to UV of the dried spores was attributable to surface characteristics (porosity and texture) of the solid materials. γ radiation was shown to accomplish similar rates of inactivation for spores in aqueous suspension and for dried spores on surfaces. Collectively, these results suggest that the application of UV or ionizing radiation may hold promise for decontamination following bioterrorism events.