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PubMedDate not supplied

Glutathione and gamma-glutamyl cycle enzymes in rat testis during sexual maturation.

The main enzymes of the gamma-glutamyl cycle in the testis were studied during the onset of spermatogenesis. The activities of gamma-glutamyl transpeptidase, 5-oxoprolinase, and gamma-glutamyl cysteine synthetase, and levels of glutathione were measured in testis homogenates and Sertoli cell preparations obtained from 10-, 18-, and 26-day-old rats. A significant increase of all enzyme activities with the animal age was observed. Level of glutathione also increased in an age-dependent manner. Since the gamma-glutamyl cycle is involved in the cellular incorporation of amino acids, the present findings suggest that this uptake mechanism may be relevant during spermatogenic onset in which synthesis and secretion of specific proteins are essential for germ cell development.

Animals↗
PubMed1989

Study of the 5-oxoprolinase reaction by 13C NMR.

5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate. Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A. P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A. P., and Meister, A. (1987) J. Biol. Chem. 262, 11020-11025). A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here. The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds. Using 5-[18O]oxo[5-13C]proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction. The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively. The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate. Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis.

Adenosine Diphosphate↗
PubMed1986

Modulation of gamma-glutamyl cycle and glutathione levels in rat mammary gland explants.

The reduced glutathione levels and the enzymes gamma-glutamyl-transpeptidase, 5-oxoprolinase and gamma-glutamylcysteine synthetase, which participate in the metabolism of glutathione through the gamma-glutamyl cycle, were determined in explants from the lactating mammary gland of the rat. Significant activities of the enzyme were found. Two modulators of this cycle were used. L-2-Oxothiazolidine-4-carboxylate, an alternative substrate of 5-oxoprolinase and an intracellular cysteine delivery system, increased the reduced glutathione levels under the experimental conditions used. The other compound utilized, DL-buthionine-SR-sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase, significantly decreased the glutathione levels. This effect is slowly reversible.

Amidohydrolases↗
PubMed1988

Interaction of the protein components of 5-oxoprolinase. Substrate-dependent enzyme complex formation.

5-Oxo-L-prolinase from Pseudomonas putida is composed of two reversibly dissociable proteins: Component A catalyzes 5-oxoproline-dependent cleavage of ATP, but does not catalyze the decyclization of 5-oxoproline; Component B is required for the coupling of ATP cleavage to ring-opening of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). We describe here the purifications of Components A and B to apparent homogeneity and the interactions between these two proteins. The cellular content of Component B activity is significantly greater than that of Component A. By gel filtration, Component A is a hexamer; but in the presence of substrates, it is a dimer. Component B can exist as an aggregate, an octamer, or a tetramer, depending upon the conditions used. Gel filtration of a mixture of Components A and B in the presence of substrates gives a unique protein species that exhibits 5-oxoprolinase activity. The Mr of this Component A-Component B complex indicates that it probably has an A2-B2 structure. The molar ratio of Component A to Component B in the complex was determined to be 1:1 by the continuous variation method (Job). Titrations of each component by the other suggest that phosphorylated 5-oxoproline-bound Component A is the entity that interacts with Component B. These studies indicate that the binding of phosphorylated 5-oxoproline-bound Component A to Component B to form a complex proceeds by a cooperative type mechanism. This is supported by the observed shifts of the intersection points of the Job curves (see Appendix).

Amidohydrolases↗
PubMed1987

18O studies on the 5-oxoprolinase reaction. Evidence for a phosphorylated tetrahedral intermediate.

5-Oxoprolinase catalyzes a reaction in which the cleavage of ATP to ADP and Pi and the decyclization of 5-oxoproline to form glutamate are coupled. When the reaction catalyzed by 5-oxoprolinase of Pseudomonas putida was carried out to 90% completion in H2(18)O, the residual 5-oxoproline was found to contain 18O in the amide carbonyl oxygen atom. Such isotopic incorporation was not observed in similar studies with a subunit of the enzyme which catalyzes 5-oxoproline-dependent ATPase and formation of a phosphorylated 5-oxoproline intermediate (Seddon, A.P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11543). When the complete reaction was carried out in H2(18)O, the products glutamate (gamma-carboxyl) and inorganic phosphate were mono- and di-labeled with 18O. Studies with 5-[18O]oxo-L-proline confirmed such replacement of the oxygen atoms of the gamma-carboxyl group of glutamate and the carbonyl oxygen of 5-oxoproline. Oxygen was not transferred from 5-oxoproline to inorganic phosphate. Studies with analogs of 5-oxoproline showed that di-labeling of inorganic phosphate occurred only when ATP hydrolysis was coupled or partially coupled with the decyclization of the substrate. Studies with 5-oxoprolinase from rat kidney gave similar results. These observations are in accord with the view that the reaction involves enzyme-bound phosphorylated intermediates and provide evidence for a phosphorylated tetrahedral intermediate, whose formation is required for coupling.

1-Carboxyglutamic Acid↗
PubMed1986

Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase.

Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.

Adenosine Triphosphate↗
PubMed1982

Interaction of 5-oxo-L-prolinase with nucleoside triphosphates. Evidence suggesting substrate-dependent conformational change.

5-Oxoprolinase catalyzes the coupled hydrolysis of ATP and 5-oxoproline to yield glutamate, ADP, and Pi; the reaction may be partially or completely uncoupled by structural modification of either substrate. In the present work, we found slow 5-oxoproline-dependent changes in the rates of hydrolysis of ITP, GTP, and UTP. For example, in the absence of 5-oxoproline, the enzyme catalyzes the hydrolysis of UTP at a rapid and constant rate. Following addition of 5-oxo-L-proline, the rate of hydrolysis decreases slowly; after about 25 min, a much slower and constant rate of hydrolysis is attained. This change in rate is associated with a decrease in Vmax and an increase in the Km for UTP. In similar studies with ATP, both Vmax and Km increase over a much shorter time period (less than 10 s). The findings indicate that 5-oxoprolinase is a hysteretic enzyme, and are consistent with the hypothesis that in the normal catalytic reaction, the binding of both ATP and 5-oxo-proline to the enzyme induces a conformational change that brings the substrates into a juxtaposition that facilitates the reaction.

Amidohydrolases↗
PubMed1982

New substrates of 5-oxo-L-prolinase.

Interaction of 5-oxo-L-prolinase (which catalyzes stoichiometric cleavage of 5-oxo-L-proline to L-glutamate coupled to that of cleavage of ATP to ADP and Pi), with a number of new 5-oxo-L-proline analogs was examined in studies in which (a) analogs were substituted for 5-oxo-L-proline and the formation of products was determined, (b) the effects of the analogs on the normal catalytic reaction were observed, and (c) the effects of the analogs on the ITPase activity exhibited by the enzyme were examined. Some of these reactions are partially coupled, i.e. the molar formation of ADP exceeds that of amino acid; others are uncoupled, i.e. cleavage of ATP, but not that of imino acid occurs. Analogs in which the 4-methylene moiety of 5-oxo-L-proline is replaced by O, S, and NH (L-2-oxooxazolidine-4-carboxylate, L-2-oxothiazolidine-4-carboxylate, and 2-imidazolidone-4-carboxylate) participate in reactions that are, partially coupled, coupled, and uncoupled respectively. Partial coupling occurs with cis- and trans-2-oxo-5-methyloxazolidine-4-carboxylate, and uncoupled reactions occur with L-2-iminothiazolidine-4-carboxylate, 2-thiazolinone-4-carboxylate, and 2-oxo-5,5-dimethylthiazolidine-4-carboxylate. These and earlier findings indicate that significant binding of the imino acid substrate requires a 5-carbonyl (or = NH), an unsubstituted N-1 and a C-2 of the L-configuration; substantial modification of 5-oxo-L-proline in the region of C-3 and C-4 is possible with retention of binding properties. Uncoupling and partial coupling may be associated with significant differences in the orientations of the analogs at the active site that facilitate (to various extents) the access of water to the nucleoside triphosphate.

Adenosine Triphosphate↗
PubMed1984

Resolution of 5-oxo-L-prolinase into a 5-oxo-L-proline-dependent ATPase and a coupling protein.

5-Oxo-L-prolinase catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic cleavage of ATP to ADP and Pi. In the present research, the enzyme present in a strain of Pseudomonas putida isolated from soil by enrichment culture was found to be composed of two protein components. Neither component alone could catalyze the 5-oxoprolinase reaction, but the reaction was effectively catalyzed when they were mixed. One component (A) exhibited 5-oxo-L-proline-dependent ATPase activity indicating that Component A can interact with both ATP and 5-oxo-L-proline. The other component (coupling protein; B) does not exhibit ATPase activity nor is there evidence that it binds 5-oxo-L-proline. The findings are consistent with (but do not prove) the hypothesis that the Component A catalyzes an initial step in the reaction which involves 5-oxoproline and ATP, such as phosphorylation of 5-oxoproline. The coupling protein (B) may function as a catalyst that converts a phosphorylated form of 5-oxoproline to glutamate, or it might alter the conformation of Component A so as to facilitate the reaction.

Adenosine Triphosphatases↗
PubMed1982

Effect of sulfhydryl group modification on the activities of 5-oxo-L-prolinase.

5-Oxo-L-prolinase was isolated from rat kidney by a new procedure; highly active and apparently homogeneous enzyme was obtained in 50% yield after 1700-fold purification. The enzyme, which couples cleavage of ATP to ADP with that of 5-oxo-L-proline to L-glutamate, is uncoupled by Ca2+, Co2+, or excess Mn2+ as well as by replacement of adenosine 5'-triphosphate or of 5-oxo-L-proline by certain analogs as previously reported. The enzyme has Mr = 325,000 and is composed of 2 apparently identical subunits. It contains 27 sulfhydryl groups/monomer, 6 of which can be titrated in the native enzyme and 2 of which are required for catalysis. One of the sulfhydryl groups that can be titrated with 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide can be protected against modification by ATP or inosine 5'-triphosphate. The findings suggest that at least 1 sulfhydryl group is at or close to the nucleoside triphosphate binding site and is involved in cleavage of NTP. 5'-p-Fluorosulfonylbenzoyl adenosine and 5'-p-fluorosulfonylbenzoyl inosine interact with the sulfhydryl group involved in NTPase activity and also with another amino acid residue of the enzyme. The data provide additional strong evidence that (a) the enzyme can bind 5-oxo-L-proline in the absence of NTP, and that it can bind NTP in the absence of 5-oxo-L-proline, and (b) the L-glutamate synthesis and NTP-cleaving activities are catalyzed by the same protein.

Adenosine Diphosphate↗
PubMed1982

Accumulation of pyroglutamic acid (5-oxoproline) in homocystinuria.

A 12-year-old boy with homocystinuria due to a cystathionine synthetase defect was shown to accumulate large amounts of pyroglutamic acid (pyroglu). In four different urine samples the concentration ranged between 4.1 and 9.7 mol/mol of creatinine. The pyroglu concentration in plasma was 1.6 mmol/l, and in erythrocytes 750 nmol/g cells. Normally only trace amounts are present. The pyroglu levels normalized during treatment with pyridoxine and folic acid. Accumulation of pyroglu has not previously been described in homocystinuria. Homocysteine can replace cysteine as a substrate for gamma-glutamylcysteine synthetase, an enzyme involved in the biosynthesis of glutathione, giving gamma-glutamylhomocysteine. This dipeptide is efficiently split by gamma-glutamylcyclotransferase to pyroglu and homocysteine. This reaction sequence most likely explains the accumulation of pyroglu in our patient. Ten other homocystinuria patients, who all had lower plasma levels of homocysteine, did not accumulate pyroglu. Our patient might either be a new variant, or more likely the pyroglu formation occurs only high levels of homocysteine.

Amino Acids↗
PubMed1994

High-resolution 1H-NMR spectroscopy of blood plasma for metabolic studies.

Although spin-echo techniques are often used to obtain 1H-NMR spectra of serum or plasma samples, they do not provide reliable quantitative analyses of metabolites. We present a standardized procedure, optimized for sensitivity, for using single-pulse 1H-NMR spectroscopy to analyze deproteinized plasma. The detection limit for various metabolites ranges between 2 and 40 mumol/L. The method allows quantitative analysis of many compounds of interest in studies of inborn errors of metabolism, including betaine and dimethylglycine, which cannot be measured easily with other techniques. For lactate, tyrosine, threonine, and alanine, we obtained results that correlated well with those obtained by established techniques. We also present a library containing resonance positions of 38 compounds occurring in plasma samples in health and disease, including 14 as-yet-unidentified resonances. As an example of the diagnostic power of the technique we show a spectrum of a plasma sample from a patient with 5-oxoprolinuria (pyroglutamic aciduria; McKusick 266130), an enzymatic defect in glutathione biosynthesis.

Blood Chemical Analysis↗
PubMed1998

Transient 5-oxoprolinuria (pyroglutamic aciduria) with systemic acidosis in an adult receiving antibiotic therapy.

5-Oxoprolinuria is a recognized condition with increased urinary excretion of 5-oxoproline and is associated with a variety of inborn metabolic defects involving the series of enzyme-linked reactions known as the gamma-glutamyl cycle. We report the unusual case of a 35-year-old woman who initially presented with staphylococcal pneumonia but went on to develop a transient high anion gap metabolic acidosis. The development and subsequent complete recovery from this acidosis were subsequently shown to be related in time to the intravenous administration of the antibiotics flucloxacillin and netilmicin. Analysis of the patient's urine for organic acids revealed massively increased excretions of 5-oxoproline at the peak of her acidosis. We suggest that this patient developed a transient disturbance in the gamma-glutamyl cycle involving the 5-oxoprolinase step, which resulted in accumulation of 5-oxoproline that caused a severe high anion gap metabolic acidosis. The administered antibiotics remain as possible causative agents.

Acidosis↗