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Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined. These enzymes were previously incorrectly annotated as adenosine deaminases or chlorohydrolases. It was shown here that these enzymes actually deaminate 6-aminodeoxyfutalosine. The deamination of 6-aminodeoxyfutalosine is part of an alternative menaquinone biosynthetic pathway that involves the formation of futalosine. 6-Aminodeoxyfutalosine is deaminated by these enzymes with catalytic efficiencies greater than 10(5) M(-1) s(-1), Km values of 0.9-6.0 μM, and kcat values of 1.2-8.6 s(-1). Adenosine, 2'-deoxyadenosine, thiomethyladenosine, and S-adenosylhomocysteine are deaminated at least an order of magnitude slower than 6-aminodeoxyfutalosine. The crystal structure of Nis0429 was determined and the substrate, 6-aminodeoxyfutalosine, was positioned in the active site on the basis of the presence of adventitiously bound benzoic acid. In this model, Ser-145 interacts with the carboxylate moiety of the substrate. The structure of Dr0824 was also determined, but a collapsed active site pocket prevented docking of substrates. A computational model of Sav2595 was built on the basis of the crystal structure of adenosine deaminase and substrates were docked. The model predicted a conserved arginine after β-strand 1 to be partially responsible for the substrate specificity of Sav2595.

Deamidation of proteins is one of the most prevalent post-translational modifications found upon aging, and in age-onset diseases. Specific asparagine and glutamine residues are often selectively deamidated during this process. In the human lens, deamidation has been shown to occur in many crystallins, but it is not clear how these deamidated proteins lead to lens opacity or cataract. Here we have modeled in vitro the effect of deamidation of specific asparagine and glutamine residues in human recombinant γS-crystallin (HGS) on the solution properties of the protein. The residues selected for deamidation in vitro are those that are found to be deamidated in aged and cataractous lenses in vivo. Two derivatives were prepared, one with Asn76 and Asnl43 deamidated (2N-HGS) and the other with two additional Gin residues (92 and 120) deamidated (2N2Q;HGS). Isoelectric focusing measurements showed the expected lowering of the pi from 6.9 in HGS to ~6.5 in 2N-HGS and to ~6.1 in 2N2Q;HGS. However, spectroscopic studies showed no significant change in the secondary and tertiary structures of the deamidated proteins relative to the wild type. The stability of 2N-HGS and 2N2Q;HGS, as measured by guanidinium hydrochloride unfolding, also remained comparable to that of HGS. The main difference was the altered protein--protein interaction among the three proteins. The net repulsive interactions that are characteristic of HGS are diminished in the deamidated derivatives as evidenced by static light scattering measurements of the second virial coefficient, B2 (B2 values for HGS, 2N-HGS, and 2N2QT1GS of 8.90 X 10-4, 7.10 × 10-4, and 6.65 × 10-4 ml. mol g-2, respectively). Further substantiation is provided by estimates of the excess binding energy of protein--protein interactions in the condensed phase, obtained from measurements of the PEG-induced liquid--liquid phase separation profiles for the three proteins. The data suggest that enhanced attractive protein--protein interactions, arising from the deamidation of HGS, promote protein aggregation, thereby leading to increased light scattering and opacity over time. [ABSTRACT FROM AUTHOR]

Deamidation is the uncatalyzed process by which asparagine or glutamine can be transformed into aspartic acid or glutamic acid, respectively. In its active homodimeric form, mammalian triosephosphate isomerase (TPI) contains two deamidation sites per monomer. Experimental evidence shows that the primary deamidation site (Asn71-Gly72) deamidates faster than the secondary deamidation site (Asn15-Gly16). To evaluate the factors controlling the rates of these two deamidation sites in TPI, we have performed graphics processing unit-enabled microsecond long molecular dynamics simulations of rabbit TPI. The kinetics of asparagine dipeptide and two deamidation sites in mammalian TPI are also investigated using quantum mechanical/molecular mechanical tools with the umbrella sampling technique. Analysis of the simulations has been performed using independent global and local descriptors that can influence the deamidation rates: desolvation effects, backbone acidity, and side chain conformations. Our findings show that all the descriptors add up to favor the primary deamidation site over the secondary one in mammalian TPI: Asn71 deamidates faster because it is more solvent accessible, the adjacent glycine NH backbone acidity is enhanced, and the Asn side chain has a preferential near attack conformation. The crucial impact of the backbone amide acidity of the adjacent glycine on the deamidation rate is shown by kinetic analysis. Our findings also shed light on the effect of high-order structure on deamidation: the deamidation in a small peptide is favored first because of the higher reactivity of the asparagine residue and then because of the stronger stability of the tetrahedral intermediate. [ABSTRACT FROM AUTHOR]

Copper amine oxidases (CAOs) are responsible for the oxidative deamination of primary amines to their corresponding aldehydes. The CAO catalytic mechanism can be divided into two half-reactions: a reductive half-reaction in which a primary amine substrate is oxidized to its corresponding aldehyde with the concomitant reduction of the organic cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and an oxidative half-reaction in which reduced TPQ is reoxidized with the reduction of molecular oxygen to hydrogen peroxide. The reductive half-reaction proceeds via Schiff base chemistry, in which the primary amine substrate first attacks the C5 carbonyl of TPQ, forming a series of covalent Schiff base intermediates. The X-ray crystal structures of copper amine oxidase-1 from the yeast Hansenula polymorpha (HPAO-1) in complex with ethylamine and benzylamine have been determined to resolutions of 2.18 and 2.25 Å, respectively. These structures reveal the two amine substrates bound at the back of the active site coincident with TPQ in its two-electron-reduced aminoquinol form. Rearrangements of particular amino acid side chains within the substrate channel and specific protein-substrate interactions provide insight into the substrate specificity of HPAO-1. These changes begin to account for this CAO's kinetic preference for small, aliphatic amines over the aromatic amines or whole peptides preferred by some of its homologues. [ABSTRACT FROM AUTHOR]

The penultimate reaction in the oxidative degradation of nicotinate (vitamin B3) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate asnidohydrolase (NicP). Although it has been considered a model system for bacterial degradation of N-heterocycic compounds, only recently have gene dusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RBSO, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from F.scherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ansmonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k~, and KM values (pH 7.S and 25°C) of 11.7 ± 0.2 s' and 128 ± 6 aM, respectively. The observed K0 of the NicF-maleate (EP) complex, also measured by JTC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of N1cF, determined at 2.4 A using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lysi 17, and CyslSO) observed m the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nudeophilic addition-elimination sequence involving the thiolate side chain of Cys 150. [ABSTRACT FROM AUTHOR]

Four proteins from NCBI cog1816, previously annotated as adenosine deaminases, have been subjected to structural and functional characterization. Pa0148 (Pseudomonas aeruginosa PAO1), AAur1117 (Arthrobacter aurescens TC1), Sgx9403e, and Sgx9403g have been purified and their substrate profiles determined. Adenosine is not a substrate for any of these enzymes. All of these proteins will deaminate adenine to produce hypoxanthine with kcat/Km values that exceed 105 M-1 s-1. These enzymes will also accept 6-chloropurine, 6-methoxypurine, N-6-methyladenine, and 2,6-diaminopurine as alternate substrates. X-ray structures of Pa0148 and AAur1117 have been determined and reveal nearly identical distorted (β/α)8 barrels with a single zinc ion that is characteristic of members of the amidohydrolase superfamily. Structures of Pa0148 with adenine, 6-chloropurine, and hypoxanthine were also determined, thereby permitting identification of the residues responsible for coordinating the substrate and product. [ABSTRACT FROM AUTHOR]

Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of l-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with l-aspartate at 2.4 and 2.6 Å resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G317SSIMPGKVN326) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate. [ABSTRACT FROM AUTHOR]

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B12), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while kcat and KMCob(I) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease. [ABSTRACT FROM AUTHOR]

The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway. [ABSTRACT FROM AUTHOR]

Coenzyme B12-dependent ethanolamine ammonia-lyase acts on both enantiomers of the substrate 2-amino-1-propanol [Diziol, P., et al. (1980) Eur. J. Biochem. 106, 211-224]. To rationalize this apparent lack of stereospecificity and the enantiomer-specific stereochemical courses of the deamination, we analyzed the X-ray structures of enantiomer-bound forms of the enzyme-cyanocobalamin complex. The lower affinity for the (R)-enantiomer may be due to the conformational change of the Valα326 side chain of the enzyme. In a manner consistent with the reported experimental results, we can predict that the pro-S hydrogen atom on C1 is abstracted by the adenosyl radical from both enantiomeric substrates, because it is the nearest one in both enantiomer-bound forms. We also predicted that the NH2 group migrates from C2 to C1 by a suprafacial shift, with inversion of configuration at C1 for both enantiomeric substrates, although the absolute configuration of the 1-amino-1-propanol intermediate is not yet known. Reported labeling experiments demonstrate that (R)-2-amino-1-propanol is deaminated by the enzyme with inversion of configuration at C2, whereas the (S)-enantiomer is deaminated with retention. By taking these results into consideration, we can predict the rotameric radical intermediate from the (S)-enantiomer undergoes flipping to the rotamer from the (R)-enantiomer before the hydrogen back-abstraction. This suggests the preference of the enzyme active site for the rotamer from the (R)-enantiomer in equilibration. This preference might be explained in terms of the steric repulsion of the (S)-enantiomer-derived product radical at C3 with the Phea329 and Leua402 residues. [ABSTRACT FROM AUTHOR]

The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5′-phosphate (PLP) enzyme DOPA decarboxylase (DDC) was here investigated. Either wild-type or Y332F DDC variant is able to perform such oxidation toward aromatic amines or aromatic L-amino acids, respectively, without the aid of any cofactor related to oxygen chemistry. Oxidative deamination produces, in equivalent amounts, a carbonyl compound and ammonia, accompanied by dioxygen consumption in a 1:2 molar ratio with respect to the products. Kinetic studies either in the pre-steady or in the steady state, together with HPLC analyses of reaction mixtures under varying experimental conditions, revealed that a ketimine accumulates during the linear phase of product formation. This species is reactive since it is converted back to PLP when the substrate is consumed. Rapid-mixing chemical quench studies provide evidence that the ketimine is indeed an intermediate formed during the first catalytic cycle. Moreover, superoxide anion and hydrogen peroxide are both generated during the catalytic cycles. On this basis, a mechanism of oxidative deamination consistent with the present data is proposed. Furthermore, the catalytic properties of the T246A DDC mutant together with those previously obtained with H192Q mutant allow us to propose that the Thr246-His192 dyad could act as a general base in promoting the first step of the oxidative deamination of aromatic amines. [ABSTRACT FROM AUTHOR]

To estimate the relative importance of alternate routes of spontaneous degradation of DNA and the rate enhancements produced by enzymes catalyzing these reactions, rate constants and thermodynamic activation parameters for the degradation of 2′-deoxynucleosides at 25 °C were determined by extrapolation of rates observed in the temperature range between 90 and 200 °C in neutral phosphate buffer. Rates of deamination of 2′-deoxycytidine, 1-methylcytosine, and cytidine were found to be identical within experimental enor (t½ ≈ 20 years, 37 °C). Rate constants for deamination of 2'-deoxyadenosine and 2′-deoxyguanosine, which could not be determined directly because of rapid glycoside cleavage, were estimated by assuming that methyl replacement should generate reasonable model substrates. The rates of deamination of 9-methyladenine and 9-methylguanine were found to be similar to each other (t½ ≈ 6000 years, 37 °C) and ∼10²-fold slower than the rates of glycoside cleavage in 2′-deoxyadenosine and 2′-deoxyguanosine. The deamination of 2′-deoxyadenosine, 2′-deoxyguanosine, and 2′-deoxycytidine led to accelerated rates of glycoside cleavage. In the exceptional case of 2′-deoxycytidine, deamination and glycoside hydrolysis proceed at very similar rates at all temperatures. Glycoside cleavage proceeds with half-times ranging from 4 years for 2′-deoxyinosine to 40 years for 2′-deoxycytidine (37 °C). The rate enhancements produced by DNA glycosylases, estimated by comparison with the rates of these uncatalyzed reactions, are found to be substantially smaller than those produced by deaminases and staphylococcal nuclease. [ABSTRACT FROM AUTHOR]

According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins, and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging. The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the βA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far-UV and near-UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to that of the wild type βA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the βA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions. [ABSTRACT FROM AUTHOR]

Asparagine deamidation is a decisive event in chemotherapy-induced apoptosis and a major obstacle in the formulation of monoclonal antibodies. Despite the importance of deamidation, little is known about the elementary reactions involved. B3LYP/6-31+G(d,p)/COSMO-RS calculations were used to obtain stable structures and transition states for a network of reactions. Calculated rate constants were incorporated into a kinetic model of the pH dependence and compared to a pseudo-steady-state model. At low pH, the calculations show that deamidation occurs by direct acid-catalyzed hydrolysis to aspartate. At neutral to basic pH, deamidation proceeds by the initial formation of a tetrahedral intermediate. The intermediate can be converted to succinimide by two pathways and three rate-determining steps that shift in relative importance with pH. The calculated pH-dependent rate constant qualitatively agrees with the experimental pH dependence. The rate-determining transition state structures may help to understand chemotherapy-induced apoptosis and improve protein formulations. [ABSTRACT FROM AUTHOR]

Although deamidation at asparagine and glutamine has been found in numerous studies of a variety of proteins, in almost all cases the analytical methodology that was used could detect only a single site of deamidation. For the extensively studied case of reduced bovine ribonuclease A (13 689 Da), only Asn67 deamidation has been demonstrated previously, although one study found three monodeamidated fractions. Here top down tandem mass spectrometry shows that Asn67 deamidation is extensive before Asn71 and Asn94 react; these are more than half deamidated before Asn34 reacts, and its deamidation is extensive before that at Gln74 is initiated. Except for the initial Asn67 site, these large reactivity differences correlate poorly with neighboring amino acid identities and instead indicate residual conformational effects despite the strongly denaturing media that were used; deamidation at Asn67 could enhance that at Asn71, and these enhance that at Gln74. This success in the site-specific quantitation of deamidation in a 14 kDa protein mixture, despite the minimal 1 Da (-NH2 → -OH) change in the molecular mass, is further evidence of the broad applicability of the top down MS/MS methodology for characterization of protein posttranslational modifications. [ABSTRACT FROM AUTHOR]

Cytotoxic necrotizing factor 1 (CNF1), a virulence factor expressed by pathogenic Escherichia coli, acts on Rho-GTPases and specifically deamidates a single glutamine residue (Gln-63 in RhoA) required for GTP hydrolysis. This modification constitutively activates the effector binding function of Rho-GTPases and eventually leads to their proteasome-mediated degradation. Previous structural investigation revealed that the CNF1 active site is located in a deep and narrow pocket and that the entrance to this pocket is formed by nine loop segments. We have examined the functional importance of five of these loops (2, 6, 7, 8, and 9) by deleting them individually. We find that deletion of proximally located loops 8 and 9 in the 32 kDa catalytic domain of CNF1 (CNF1-C) nearly or completely abolishes deamidation of RhoA in vitro, identifying a potential Rho-GTPase recognition site. Deletion of loop 7 causes protein folding errors, and deletion of loop 6 has a small effect on deamidation. In contrast, deletion of loop 2 is found to increase deamidation 5-7-fold, implying that this loop rearranges in binding RhoA. None of the loop deletions or wild-type CNF1-C is able to deamidate RhoA containing Asn-63 instead of Gln-63, suggesting that the fit between the toxin and its target is highly precise. In addition, we show that the specificity constant (mcat/Kin) of CNF1-C for RhoA is 825 ± 3 M[SUP-1] s[SUP-1]. This modest value is consistent with the confining size of the active site pocket acting to exclude nonspecific targets but also limiting reactivity toward intended targets. [ABSTRACT FROM AUTHOR]

Studies the oxidative deamination reaction catalyzed by Clostridium symbiosum glutamate dehydrogenase producing the real-time reaction courses. Assumption on the basis of steady-state kinetics; Mechanisms of the static enzyme complexes; Conversion of fluorescence intensities to micromolar concentrations.

Examines the deamidation among disulfide bonded ΓS-crystallins from cataractous lenses. Association of deamidation with cataract; Insolubility of crystallins to opacity; Use of ion exchange chromatography.

Examines the mutations of endonuclease V from Thermotoga maritima. Involvement of the residues in coordinating catalytic metal ions; Role of K139A in facilitating non-inosine substrate cleavage; Acceleration of adenine deamination in high temperatures.

Reports on the measurement of [sup 15]N isotope effects and solvent deuterium isotope effects for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. Isotope effect of mutant His102Asn; Estimation of the forward commitment of the reaction with the wild-type enzyme.

Investigates the regioselectivity and steady-state kinetics of tissue transglutaminase (tTGase)-catalyzed deamidation of known immunogenic peptides to dissect the molecular recognition features of tTGase for gluten derived products. Provision of a quantitative framework for understanding the role of tTGase in Celiac Sprue, an autoimmune disorder of the human small intestine; Kinetic analysis of tTGase.

Investigates complexes formed by D145N dehalogenase by analyzing the role played by Aspartate 145 in the active site of 4-chlorobenzoyl-CoA dehalogenase. Possibility that the product bound to the active site of asparagine 145 dehalogenase catalyzes the deamidation of the asparagine side chain to produce the wild-type aspartate 145.

Demonstrates the isolation of a sulfolobus solfataricus (SsADH). Preparation of the mutant enzyme from Escherichia coli to homogenous form; Determination of kinetic thermal stabilities; Thermal unfolding of the enzyme; Deamidation during thermoinactivation; Alcohol dehydrogenase from the archeon SsADH; Expression and location of the mutation.

Presents an experiment determining the extents of deamidation of glutamine-50, glutamine-6, and glutamine 147 from alpha-A crystallin found in the fetal-embryonic regions of lenses from different age donors. Methodology used to conduct the experiment; Information on the mechanisms responsible for the low rates of glutamine deamidation in vivo; Findings of the experiment.

Presents a study which concluded that adenosine deaminase (ADA) which catalyzes the hydrolytic deamination of adenosine nucleosides to inosine nucleosides and ammonia is an important enzyme in purine metabolism and lymphoid development. How the x-ray structures of murine ADA were determined; Information on ADA; Details on the experiments conducted.

Provides information on a study which demonstrated that the dopa decarboxylase (DDC) reaction to alpha-methyldopa, leads to oxidative deamination which produced 3,4-dihydroxyphenylacetone and ammonia. What DDC catalyzes; Identification of a ketone; Methodology used to conduct the study; Results of the study.

Focuses on the deamidation of recombinant human stem cell factor during aging. Information on the deamidation rate; What cell proliferation assays have shown; Indication of results; Initial expression of deamidation molecule.

Examines the rate of ultra-violet-induced deamination of cystosine to uracil at a specific site in a double-stranded DNA. Finding that the rate of appearance of tandem double mutations during incubation of UV-irradiated DNA is inconsistent with two random and independently occurring mutational events.

Discusses the identification of substrate analogue L-aspartase `beta'-semialdehyde (L-ASA) as a mechanism-based inactivation of L-aspartase from Escherichia Coli. Description of materials and methods of study; Results of study; Discussion of results; References.

Burst-phase kinetic analysis was used to evaluate the deamination rate of the aminated–methylidene imidazolone (NH2–MIO) adduct of a Taxus phenylalanine aminomutase. The kinetic parameters were interrogated by a non-natural substrate (S)-styryl-a-alanine that yielded a chromophoric styrylacrylate product upon deamination by the aminomutase. Transient inactivation of the enzyme by the NH2–MIO adduct intermediate resulted in an initial burst of product, with reactivation by deamination of the adduct. This study validated the rate constants of a kinetic model demonstrating that the NH2–Mb adduct and cinnamanate intermediate are sufficiently retained to catalyze the natural α- to β-phenylalanine isomerization. [ABSTRACT FROM AUTHOR]

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: kcat = 49 s-1, Km = 72 μM, and kcat/Km = 6.7 × 105 M-1 s-1. The kinetic constants for the deamination of cytosine are as follows: kcat = 45 s-1, Km = 302 μM, and kcat/Km = 1.5 × 105 M-1 s-1. Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site. [ABSTRACT FROM AUTHOR]

The methylation of cytosine in the full mutation of the expanded CGG repeat and subsequent deamination to thymine could be a measure of repeat instability. We report the synthesis of NCD-Bpy, which binds to the TGG/CGG site in the repeat hairpin. NCD-Bpy forces the thymine in the TGG/CGG site to flip out from the π-stack, recruits osmium tetroxide in the vicinity of the flipped-out T, and oxidizes the T. The piperidine-induced cleavage band successfully determined the position of the T in the expanded CGG repeat.