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  • HYDROLYSIS AND TRANSFER REACTIONS CATALYZED BY w-AMIDASE PREPARATIONS *

    BY ALTON MEISTER, LEON LEVINTOW, ROBERT E. GREENFIELD, AND PATRICIA A. ABENDSCHEIN

    (From the Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland)

    (Received for publication, December 22, 1954)

    Recent studies have shown that deamidation of glutamine and aspara- gine may take place by at least several types of enzymatic reactions. These include (a) hydrolysis of glutamine or asparagine to the respective or-aminodicarboxylic acids and ammonia (1, 2), (b) hydrolysis as in (a) but dependent upon the presence of phosphate or certain other anions for activity (3-5), (c) deamidation of glutamine and asparagine by mecha- nisms involving participation of an a-keto acid (6-9), (d) deamidation of glutamine to glutamate associated with the synthesis of adenosine tri- phosphate from adenosine diphosphate and phosphate (i.e., reversal of gluta.mine synthesis) (lo), and (e) deamidation of glutamine and aspara- gine associated with w replacement reactions (11-16). Studies in this lab- oratory have shown that the deamidation reactions described under (c) are associated with transaminat.ion leading to formation of the a-amino acid analogous to the cy-keto acid (8,9). The formation of ammonia in the ar-keto acid-dependent deamidation reactions may be ascribed to enzymatic hydrolysis of the oc-keto acid w-amides, a-ketoglutaramic and cr-ketosuccin- amic acids, which appear to be intermediates in these reactions. Extracts of a number of tissues catalyze the hydrolysis of the.&-keto acid w-amides, and a purified enzyme preparation capable of catalyzing these reactions has been obtained from rat, liver (17, 18).

    The present communication describes studies on certain properties of this liver enzyme as well as those of preparations of a bacterial glutaminase and guinea pig serum asparaginase. The liver enzyme catalyzes the hy- drolysis of or-ketosuccinamic, cu-ketoglutaramic, succinamic, and glutaramic acids, but does not deamidate glutamine or asparagine. It also catalyzes the formation of succinylmonohydroxamic and glutarylmonohydroxamic acids from the corresponding amides and hydroxylamine much more rapidly than the hydrolysis of these amides. On the other hand, the glutaminase and asparaginase preparations studied here catalyze hydroxamic acid for- mation from glutamine and asparagine, respectively, at rates relatively

    *Presented in part before the Division of Biological Chemistry at the 127th meeting of the American Chemical Society at Cincinnati, April 1, 1955.

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  • 442 w-AMIDASE PREPARATIONS

    low compared to those of hydrolysis. It is of interest that, in the presence of hydroxylamine, all of these preparations also catalyze monohydroxamic acid synthesis from the dicarboxylic acids corresponding to the susceptible amides. The findings are discussed in terms of the concept that the trans- fer and hydrolysis reactions are catalyzed by the same enzyme.

    EXPERIMENTAL

    Substrates-The following compounds were prepared as described : bar- ium a-ketoglutaramate (17)) sodium cY-ketosuccinamate (17)) sodium (Y- ketoadipamate (18)) barium cr-keto-dl-y-methylglutaramate (18)) D- and L-glutamine (lo), D- and L-homoglutamine (18)) cY-methyl-nn-glutamine (18)) dl-y-methyl-n-glutamine (18)) adipamide (19)) adipamic acid (20)) glutaramic acid (20), glutaramide (19), oxamic acid (21), L-y-glutamyl- hydroxamic acid (lo), succinamic acid,l sodium malonamate,l potassium succinylhydroxamate (22).

    Acetamide, propionamide, valeramide, isovaleramide, caproamide, and malonamide were obtained from the Eastman Kodak Company. Bu- tyramide, isobutyramide, oxamide, and succinamide were Kahlbaum prod- ucts. n-Asparagine monohydrate ( [OI]~~ - 29.2°)2 and L-asparagine mono- hydrate ([O(]:~ +29.2”)z were obtained from the Nutritional Biochemicals Corporation. Several of the commercial preparations required recrystal- lization; all of the compounds gave theoretical values for nitrogen3

    The authors are indebted to Dr. Jesse P. Greenstein, Dr. Sanford M. Birnbaum, and Dr. Milton Winitz of this Laboratory for the amino acid a-amides used in these studies, to Dr. Karl Pfister of Merck and Company for the ar-methyl-nn-asparagine, and to Dr. Simon Black of the National Institute of Arthritis and Metabolic Diseases for a sample of L-P-aspartyl- hydroxamic acid.

    dl-fi-Methyl-DL-glutamic Acid-This compound was prepared from di- ethylacetamidomalonate and ethyl crotonate by a procedure analogous to those of Snyder et al. (23) and Done and Fowden (24) for glutamic and y-methylglutamic acids, respectively. 3.6 gm. of sodium were added to. 600 ml. of absolute ethanol, and, after the reaction was complete, 326 gm. of diethylacetamidomalonate were added and the mixture was boiled under a reflux. 208 gm. of ethyl crotonate were added dropwise over a 5 hour period, and boiling was continued for an additional 12 hours. Evapora- tion of the solvent yielded a white crystalline compound, which was boiled

    1 Otani, T. T., and Meister, A., Abstracts, Division of Biological Chemistry, American Chemical Society, 127th meeting, Cincinnati, April I (1955).

    2 9 per cent, in 3 N hydrochloric acid. 3 The microanalpses were carried out by Dr. William C. Alford and Mr. Robert J.

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  • MEISTER, LEVINTOW, GREENFIELD, AND ABENDSCHEIN 443

    under a reflux with 10 times its weight of 6 N hydrochloric acid for 18 hours. The product was isolated and crystallized as described for y-methylglu- tamic acid (18). Calculated for C6H1104N: C 44.7, H 6.9, N 8.7; found, C 44.3, H 6.8, N 8.8.

    dl-~-Methyl-~~-glutamine-Carbobenzoxy-dl-~-methyl-n~-glutamic acid diamide was prepared and found to be resistant to deamidation by papain; the w-amide was therefore prepared via the carbobenzoxy w ester as de- scribed previously for a-methyl-nn-glutamine (18). Calculated for d&/3- methyl-nn-glutamic acid y-ethyl ester, CsH1604N: C 50.7, H 7.9, N 7.4; found, C 50.4, H 8.2, N 7.6. Amidation of carbobenzoxy-dl-fi-methyl-nn- glutamic acid y-ethyl ester proceeded more slowly than did amidation of the corresponding cr-methylglutamic and a-aminoadipic acid derivatives; amidation of the ,&methyl compound was complete after 4 days at room temperature. Calculated for d@-methyl-nn-glutamine, C6H1203N2: C 45.0, H 7.6, N 17.5; found, C 44.9, H 7.7, N 17.3.

    dl-a-Aminomalonamic Acid-80 gm. of bromine were added dropwise over a 3 hour period to 66 gm. of malonic acid-monoethyl ester dissolved in 250 ml. of dry ether. The mixture was kept at about 25” by occasional use of an ice bath. After all the bromine was added, the mixture was stirred for an additional 30 minutes. The ether and hydrogen bromide were removed in vacua, and the crude sirupy bromo compound was cooled to -10” and added to 1 liter of cold 28 per cent aqueous ammonia. The mixture was allowed to stand for 1 day in a stoppered bottle at room tem- perature, following which the volume was reduced to about 200 ml. by evaporation in vacua. 1 liter of a 20 per cent solution of lead acetate was added, and, after standing for several hours, the precipitated lead salt was filtered and washed with cold water until the wash water was free of halogen and ammonia. The lead salt was decomposed with hydrogen sulfide, and about 30 gm. of crude ol-aminomalonamic acid were obtained by evap- oration of the solution to dryness in vacua. Paper chromatography re- vealed that the product was contaminated with glycine, glycine amide, and aminomalonic acid. Attempts to recrystallize the product from warm (50”) water resulted in conversion of the desired amide to glycine and glycine amide, and some breakdown was observed, even at room tempera- ture. Purification was carried out by chromatography on an Amberlite XE-64 column in the acid form (170 X 2.5 cm.) as follows. 2 gm. of crude material dissolved in 10 ml. of water (pH 5.2) were added to the top of the column, and elution was carried out with water, a flow rate of 0.67 ml. per minute being used. Sixty fractions of 10 ml. each were collected. Tubes 26 to 30 contained ar-aminomalonic acid and a brown material which did not give a ninhydrin reaction. Tubes 31 to 34 apparently contained only a-aminomalonamic acid. Tubes 35 to 37 did not give a ninhydrin

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  • 444 w-AMIDASE PREPARATIONS

    reaction, Tubes 38 to 45 contained glycine, and glycine amide appeared in later fractions. The fractions containing the product were combined and lyophilized. 850 mg. of a light white powder were obtained, which gave the following analyses. Calculated for C3H603N2: C 30.5, H 5.1, N 23.7; found, C 30.1, H 5.3, N 23.7. The product was stable in aqueous solution at room temperature for only a few hours, after which time glycine amide formation was detected. When such solutions were heated at 100’ for 2 hours, complete destruction of the product with conversion to glycine amide, glycine, and ammonia was observed. Treatment with 2 N hydro- chloric acid at 100” for 2 hours yielded glycine and stoichiometric quantities of ammonia.

    Procedures-In the course of purification of the Escherichia coli glu- taminase, assays were performed as foll

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