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.     

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.     

Role of lysine 39 of alanine racemase from Bacillus stearothermophilus that binds pyridoxal 5'-phosphate. Chemical rescue studies of Lys39 --> Ala mutant

The lysine residue binding with the cofactor pyridoxal 5'-phosphate (PLP) plays an important role in catalysis, such as in the transaldimination and abstraction of alpha-hydrogen from a substrate amino acid in PLP-dependent enzymes. We studied the role of Lys39 of alanine racemase (EC 5.1.1.1) from Bacillus stearothermophilus, the PLP-binding residue of the enzyme, by replacing it site-specifically with alanine and characterizing the resultant K39A mutant enzyme. The mutant enzyme turned out to be inherently inactive, but gained an activity as high as about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine. The amine-assisted activity of the mutant enzyme depended on the pKa values and molecular volumes of the alkylamines used. A strong kinetic isotope effect was observed when alpha-deuterated D-alanine was used as a substrate in the methylamine-assisted reaction, but little effect was observed using its antipode. In marked contrast, only L-enantiomer of alanine showed a solvent isotope effect in deuterium oxide in the methylamine-assisted reaction. These results suggest that methylamine serves as a base not only to abstract the alpha-hydrogen from D-alanine but also to transfer a proton from water to the alpha-position of the deprotonated (achiral) intermediate to form D-alanine. Therefore, the exogenous amine can be regarded as a functional group fully representing Lys39 of the wild-type enzyme. Lys39 of the wild-type enzyme probably acts as the base catalyst specific to the D-enantiomer of alanine. Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the K39A mutant.     

Inactivation of cysteine proteases

The cysteine proteases papain and cathepsin B are inactivated by a Michael acceptor, a peptidyl-beta-chloro-alpha, beta-unsaturated ester (N-Ac-L-Phe-NHCH2-CCl=CH-COOMe). Inactivation occurred concomitant with chloride release which was stoichiometric with the amount of enzyme. This result is consistent with nucleophilic attack of the active site cysteine on the beta-carbon of the inhibitor, followed by expulsion of chloride ion. Inactivation by this class of compounds requires the carbon skeleton about the double bond to be in the trans configuration. The cis isomer was a competitive inhibitor. The difference in the mode of inhibition between the isomers is probably due to non-productive binding of the cis isomer due to bulky chlorine substituent in the beta-position.     

Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.     

Mutational analysis of human uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (URO-D), a heme biosynthetic enzyme, catalyzes the multi-step decarboxylation reaction converting uroporphyrinogen I or III to coproporphyrinogen I or III. The URO-D protein has been purified from several sources and its gene has been cloned from many organisms. In spite of this, little is known about the active site(s) of the enzyme. Inhibitor studies suggest that cysteine and histidine residues are important for enzyme activity. We employed the Kunkel method of site-directed mutagenesis to convert each of the six cysteines in human URO-D to serine and each of the three conserved histidines to asparagine. Recombinant mutant URO-D's were expressed in Escherichia coli, partially purified, and their kinetic properties compared to recombinant wild-type URO-D. All cysteine mutants retained approx. 40% wild-type enzyme activity, indicating that no single cysteine is absolutely critical for the integrity of the catalytic site. The three histidine mutants also retained significant enzyme activity and one, (H339N), displayed unique properties. The H339N mutation resulted in an enzyme with high residual activity but decarboxylation of intermediate reaction products of the I isomer series was markedly abnormal. The histidine at residue 339 is likely important in imparting isomer specificity.     

Study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with an enzyme-generated affinity alkylator: dual enzyme activity at a single active site

The substrate 17 beta-[(1S)-1-hydroxy-2-propynyl]-androst-4-en-3-one (beta-HPA) and its enzyme-generated alkylating product 17 beta-(1-oxo-2-propynyl)androst-4-en-3-one (OPA) were synthesized to investigate the relationship between the 3 alpha and 20 beta activities observed in commercially available cortisone reductase (EC 1.1.1.53) from Streptomyces hydrogenans. beta-HPA, a substrate [apparent Km = 145 microM; Vmax = 63 nmol (min microgram)-1], when enzymatically oxidized by cortisone reductase of OPA, inactivates simultaneously the 3 alpha and 20 beta activities in a time-dependent and irreversible manner following pseudo-first-order kinetics. OPA alone, an affinity alkylating steroid (KI = 40.5 microM; k3 = 1.8 X 10(-2) S-1), simultaneously inactivates 3 alpha and 20 beta activities in a time-dependent and irreversible manner. At pH 7, the t 1/2 of enzyme inactivation for beta-HPA (10 h) or OPA (41 min) is slower than at pH 9.2 (beta-HPA, 16 min, and OPA, 3.3 min). Substrates (progesterone, 20 beta-hydroxypregn-4-en-3-one, and 5 alpha-dihydrotestosterone), but not all steroids (20 al]ha-delta 4-pregn-4-en-3-one and 17 beta-estradiol), protect against loss of both enzyme activities by beta-HPA and OPA. The alpha isomer of HPA is not enzymatically oxidized and therefore does not cause inactivation of either 3 alpha or 20 alpha activity. Thus, beta-HPA functions as a substrate for the enzymatic generation of a powerful affinity alkylator of cortisone reductase. Second, the identical change in both the 3 alpha and 20 beta activities in all experimental conditions clearly results from dual enzyme activity at a single enzyme active site.     

Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids

Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not of L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer of beta-fluoroalanine for alpha, beta-elimination of HF to yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis to pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) and suicidal enzyme alkylation by covalent modification with an average partition ratio of 40-60 turnovers/inactivation event/monomer unit of this tetrameric enzyme. Enzyme inactivation occurs with stoichiometric incorporation of radioactive label from D-[1,2-14C]fluoroalanine. Titration of enzymic cysteinyl --SH groups with 5,5'-dithiobis(2-nitrobenzoate) indicates loss of 1 --SH group on inactivation. Acid hydrolysis of radioactive-inactive enzyme confirms cysteine residue modification. Treatment of inactive enzyme with 6 M urea, then KBH4, followed by acid hydrolysis yields two radioactive compounds, lanthionine and S-carboxyhydroxyethylcysteine, in about equal amounts. The addition of tetrahydrofolate stimulates both pyruvate production and inactivation to equal extents with about a 200-fold rate acceleration at 0.5 mM tetrahydrofolate to turnover numbers of approximately 120 min-1. The Km for D-fluoroalanine is high, 10-60 mM, and this low substrate affinity suggests D-fluoroalanine will not be a useful in vivo agent for selective inactivation of liver cell serine transhydroxymethylases.     

Elevated muscle citrate does not reduce carbohydrate utilization during tetanic stimulation

The lysosomal cysteine proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-beta, TNF-alpha, and IL-1 beta. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-beta, TNF-alpha, and IL-1 beta, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastase at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor complex. In addition, our data from neutrophil elastase activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo.     

Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair

Eukaryotic DNA polymerase beta (pol beta) can catalyze DNA synthesis during base excision DNA repair. It is shown here that pol beta also catalyzes release of 5'-terminal deoxyribose phosphate (dRP) residues from incised apurinic-apyrimidinic sites, which are common intermediate products in base excision repair. The catalytic domain for this activity resides within an amino-terminal 8-kilodalton fragment of pol beta, which comprises a distinct structural domain of the enzyme. Magnesium is required for the release of dRP from double-stranded DNA but not from a single-stranded oligonucleotide. Analysis of the released products indicates that the excision reaction occurs by beta-elimination rather than hydrolysis.     

Mechanistic consequences of mutation of active site carboxylates in a retaining beta-1,4-glycanase from Cellulomonas fimi

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Br?nsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.     

Purification of active calpain by affinity chromatography on an immobilized peptide inhibitor

The 19F-NMR resonance of 5-[19F]fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rate 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine. Comparison of pKa values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M. Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and kcat has been reduced by a factor of 10(8). 5-[19F]Fluoropyrimidin-2-one ribonucleoside is strongly fluorescent, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (Kd = 4.4 x 10(-8) M) than does the native enzyme (Kd = 1.1 x 10(-7) M). 19F-NMR indicates, however, that the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state. These results are consistent with a major role for Glu-104, not only in stabilizing the ES++ complex in the transition state, but also in destabilizing the ES complex in the ground state.     

Monomers of human beta 1 beta 1 alcohol dehydrogenase exhibit activity that differs from the dimer

A previously unreported enzymatic activity is described for monomers of the beta 1 beta 1 isoenzyme of human alcohol dehydrogenase that were prepared from dimeric enzyme by freeze-thaw in liquid nitrogen. Whereas the dimeric enzyme has optimal activity at low substrate concentrations (2.5 mM ethanol, 50 microM NAD+; "low Km" activity), the monomer has its highest activity at high substrate concentrations (1.5 M ethanol, 2.5 mM NAD+; "high Km" activity). While the activity of the monomer does not appear to be saturated at 1.5 M ethanol, its maximal activity at this high ethanol concentration exceeds the Vmax of the dimer by about 3-fold. The apparent Km of NAD+ with monomers is 270 microM, and no activity could be detected with nicotinamide mononucleotide as cofactor. During gel filtration the high Km activity elutes at a lower apparent molecular weight position than the dimer. The kinetics of monomer-to-dimer reassociation are consistent with a second-order process with a rate constant of 240 M-1 s-1. The reassociation rate is markedly enhanced by the presence of NAD+. During refolding of beta 1 beta 1 following denaturation in 6 M guanidine hydrochloride, an enzyme species with high Km activity and spectral properties similar to the freeze-thaw monomer is observed, indicating that a catalytically active monomer is an intermediate in the refolding pathway. The enzymatic activity of the monomer implies that the intersubunit contacts of beta 1 beta 1 are not crucial in establishing a catalytically competent enzyme. However, the differences in specific activity and Km between monomer and dimer suggest that dimerization may serve to modulate the catalytic properties.     

Steroid glucuronyltransferases of rat liver. Properties of oestrone and testosterone glucuronyltransferases and the effect of ovariectomy, castration and administration of steroids on the enzymes

1. Microsomal preparations from rat liver, kidney and intestine were tested for UDP-glucuronyltransferase activity by using oestrone, oestradiol-17 beta, oestriol, testosterone, cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone as substrates. The microsomal preparation from the liver glucuronidated oestrone, oestradiol-17 beta and testosterone. 2. The specific activity of the enzyme was significantly higher in livers from female rats than in those from male rats. 3. Testosterone was actively glucuronidated by both sexes. Cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone were not glucuronidated by any of the three tissues. 4. The non-ionic detergent Lubrol WX activates liver microsomal UDP-glucuronyltransferase 2-3-fold with oestrone and testosterone as substrates. 5. Oestrone glucuronyltransferase was inhibited by oestradiol-17 beta, predominantly competitively and by testosterone non-competitively. Bilirubin was a non-competitive inhibitor of oestrone glucuronidation. p-Nitrophenol had no effect. 6. Oestrone glucuronyltransferase could not be stimulated by either acute or prolonged treatment of animals with phenobarbital, whereas a single dose of 3-methylcholanthrene led to a moderate stimulation. 7. Ovariectomy leads to a 56% decrease in oestrone glucuronyltransferase activity; administration of oestradiol-17 beta induces the enzyme to normal activity after 12 days, and after 15 days the activity is twice the control value. Actinomycin D and cycloheximide block the oestradiol-17 beta-induced increase in enzyme activity. 8. Castration has no effect on the activity of testosterone glucuronyltransferase, nor does administration of testosterone influence enzyme activity. The results provide strong evidence for the existence of multiple steroid glucuronyltransferases in the liver of the rat.     

Inhibition of pigeon breast muscle alpha-ketoglutarate dehydrogenase by structural analogs of alpha-ketoglutarate

Inhibition of alpha-ketoglutarate dehydrogenase (KGD) by dicarboxylates with (oxaloacetate and ketomalonate) and without (malonate, succinate, and glutarate) alpha-keto group was studied. Ketodicarboxylates at low concentrations inhibit KGD in competitive manner. Increase in their concentrations results in appearance of the noncompetitive component. The extent of KGD inhibition by keto dicarboxylates increases with structural similarity of the inhibitor and the substrate, irrespective of preliminary incubation of the enzyme with the inhibitor. This is indicative of blocking the substrate-binding site of KGD by dicarboxylates. In contrast, inhibitory effect of dicarboxylates which contain no keto group increases as their structural similarity with the substrate decreases. Saturation of KGD with dicarboxylates of this type does not completely suppress the enzymatic activity. Alternatively, these analogs display competitive mode of inhibition. Analysis of the data obtained suggests that these dicarboxylates produce catalytically active triple complex keto substrate-KGD-dicarboxylate and that KGD which enters the composition of such a complex inhibits a decreased affinity for the keto substrate as a result of the inhibitor binding.     

Characterization of alpha-ketoglutarate-dependent taurine dioxygenase from Escherichia coli

The Escherichia coli tauD gene is required for the utilization of taurine (2-aminoethanesulfonic acid) as a sulfur source and is expressed only under conditions of sulfate starvation. The sequence relatedness of the TauD protein to the alpha-ketoglutarate-dependent 2,4-dichlorophenoxyacetate dioxygenase of Alcaligenes eutrophus suggested that TauD is an alpha-ketoglutarate-dependent dioxygenase catalyzing the oxygenolytic release of sulfite from taurine (van der Ploeg, J. R., Weiss, M. A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M. A., and Leisinger, T. (1996) J. Bacteriol. 178, 5438-5446). TauD was overexpressed in E. coli to approximately 70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure. The apparent Mr of 81,000 of the native protein and the subunit Mr of 37,400 were consistent with a homodimeric structure. The pure enzyme converted taurine to sulfite and aminoacetaldehyde, which was identified by high pressure liquid chromatography after enzymatic conversion to ethanolamine. The reaction also consumed equimolar amounts of oxygen and alpha-ketoglutarate; ferrous iron was absolutely required for activity; and ascorbate stimulated the reaction. The properties and amino acid sequence of this enzyme thus define it as a new member of the alpha-ketoglutarate-dependent dioxygenase family. The pure enzyme showed maximal activity at pH 6.9 and retained activity on storage at -20 degrees C for several weeks. Taurine (Km = 55 microM) was the preferred substrate, but pentanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 1,3-dioxo-2-isoindolineethanesulfonic acid were also desulfonated at significant rates. Among the cosubstrates tested, only alpha-ketoglutarate (Km = 11 microM) supported significant dioxygenase activity.     

Nuclear magnetic resonance studies of D2O-substrate exchange reactions catalyzed by glutamic pyruvic and glutamic oxaloacetic transaminases

Nuclear magnetic resonance studies in D2O (greater than 90%) with glutamic pyruvate transaminase (GTP) (2.6.1.2) demonstrate that this enzyme catalyzes the rapid exchange of both the alpha and beta hydrogens of L-alanine, the exchange of only one alpha hydrogen of glycine, and the beta hydrogens of pyruvate and fluoropyruvate. When the beta hydrogens of L-alanine undergo the enzyme-catalyzed exchange, the product may have 1, 2 or 3 of beta hydrogens exchanged. The exchange is stimulated by the addition of catalytic amounts of copartner of transaminations reaction. A mechanism is proposed for an extension of the conjugated system to include the alpha and beta carbons to explain the labilization of the beta hydrogens.     

Neck sprain in patients injured in car accidents: a retrospective study covering the period 1970-1994

Vigabatrin (gamma-vinyl GABA) is an antiepileptic drug and blocks GABA transaminase activity resulting in elevations in cellular GABA levels in the brain. Nipecotic acid (NPA) promotes release of GABA from neonatal optic nerve astrocytes, resulting in a bicuculline-sensitive depolarization of the optic nerve axons. The NPA-induced depolarization of vigabatrin-treated rats (100 mg/kg, i.p.) more than doubled, suggesting an elevation in free GABA levels; the GABA transporter inhibitor, NO-711 reduced the depolarization. These results are consistent with the known ability of vigabatrin to block the GABA degradation enzyme GABA-transaminase, suggesting that vigabatrin elevates astrocytic GABA levels, thereby favoring greater release of GABA through the GABA transporter.     

Utilization of glass HPLC autosampler vials as cuvettes in an iodometric assay of hydroperoxides

Bromide treatment was successful in controlling seizures in an 11-year-old Dachshund with epilepsy and presumptive phenobarbital-associated hepatopathy. Because bromide does not induce liver enzyme activity and does not seem to be hepatotoxic, it can be used to control seizures in dogs with concurrent epilepsy and hepatic disease. In this dog, institution of a special calculolytic diet with high chloride content was associated with a decrease in serum bromide concentrations and the recurrence of seizures. High chloride intake increases the elimination of bromide in dogs, leading to higher dosage requirements for bromide in dogs fed high-chloride diets.