GLUTAMATE DECARBOXYLASE STEREOCHEMISTRY VOL 17, NO. zyxw 4, 1978 669 Stereochemistry of Reactions Catalyzed by Glutamate Decarboxylase? Hidenori Yamada and Marion H. O’Leary* ABSTRACT: When the decarboxylation of L-glutamic acid by the glutamate decarboxylase from Escherichia coli is car- ried out in D20, the product y-aminobutyric acid contains a single deuterium atom. The stereochemistry of this material was established by conversion to levorotatory methyl 4- phthalimid0[4-~H] butyrate. The dextrorotatory isomer of the latter compound was synthesized from S-[2-2H]glycine by a S t u d i e s of the stereochemistry of reactions catalyzed by pyridoxal-P dependent enzymes have begun to provide a con- sistent picture for a variety of enzymes (Dunathan, 1971). These reactions generally proceed first by formation of a Schiff base between enzyme-bound pyridoxal-P and the amino acid substrate, followed by cleavage of one of the bonds to the zyxwvu a carbon of the amino acid, forming a quinoid intermediate. It has been suggested (Dunathan & Voet, 1974) that all bond- making and breaking steps may occur on the same face of this quinoid intermediate, and cases studied to date seem to bear out this suggestion. Glutamate decarboxylase (EC 4. I. I. 15) requires pyridox- al-P for activity. Decarboxylation of glutamic acid in D20 results in incorporation of a single atom of deuterium into the product y-aminobutyric acid (Mandeles et al., 1954; Yamada & O’Leary, 1977) and the enzyme does not catalyze hydrogen exchange between this product and the solvent (Yamada & O’Leary, 1977). The enzyme also catalyzes the decarboxyla- tion of a-methylglutamic acid (Huntley & Metzler, 1967; Sukhareva & Torchinsky, 1966) at a rate which is approxi- mately 600 times slower than the rate of decarboxylation of L-glutamic acid (Yamada & O’Leary, unpublished). In ap- proximately 1% of decarboxylations of a-methylglutamic acid the reaction products are y-ketovaleric acid and pyridox- amine-P as a result of transamination following decarboxyl- ation (Huntley & Metzler, 1967; Yamada & O’Leary, un- published). The stereochemistry of the tritiated pyridoxami- ne-P formed when this decarboxylation is carried out in tri- tiated water has been determined by Sukhareva et al. (1972). Although the stereochemistry of two other pyridoxal-P de- pendent enzymatic decarboxylations has been determined (Belleau & Burba, 1960; Leistner & Spenser, 1975), the stereochemistry of the decarboxylation of glutamic acid and of a-methylglutamic acid has not been determined. The latter result is of particular interest because of the existence of the decarboxylation-dependent transamination. Experimental Section General. All chemicals were the highest purity available. Glutamate decarboxylase was prepared as described previously (O’Leary, 1969). Serine transhydroxymethylase was provided by Dr. L. V. Schirch. Mass spectra were obtained with an From the Department of Chemistry, University of Wisconsin, Mad- ison, Wisconsin zyxwvutsrqponml 53706. zyxwvutsrqponm Receioed zyxwvutsrqp August IO, 1977. This research was supported by Grant NS-07657 from the National Institutes of Health. 0006-2960/78/0417-0669$01 .OO/O series of reactions not affecting the stereochemistry at the chiral center. Thus, the decarboxylation of glutamic acid OC- curs with retention of configuration. Decarboxylation of L- a-methylglutamic acid by this enzyme produces levorotatory y-aminovaleric acid and thus also occurs with retention of configuration. MS-9 mass spectrometer, N M R spectra with a Jeol MH-100, and optical rotations were measured with a Durrum-Jasco Model 5-20 spectropolarimeter. (R)-(-)-4-Amin0[4-~H]butyric Acid. To 100 mL of D20 (99.8 atom % D) was added 3 mg of solid glutamate decar- boxylase, followed by 100 mg of pyridoxal-€’, 50 mg of di- thiothreitol, 1 g of monosodium L-glutamate, and 1 g of L- glutamic acid. Over a 3-day period the mixture was stirred at 20 zyxwvu OC and a total of IO g of L-glutamic acid was added. The pD was maintained at approximately 4.5 by occasional addi- tion of DCI. The enzyme was removed by precipitation with trichloroacetic acid and centrifugation, after which the solution was lyophilized. The residue was redissolved and chromato- graphed on Amberlite XE 64 (H+ form, 2 X 75 cm). Un- reacted glutamic acid was eluted with water, and then (R)- (-)-4-amin0[4-~H]butyric acid was eluted with a gradient of 1 L of water and 1 L of 0.5 N acetic acid. The product was lyophilized and recrystallized from ethanol-water, mp 203-204 OC (dec) (lit. 202 O C (dec); Tafel & Stern, 1900). A small portion of this material was converted by pyrolysis to 4-butyrolactam (Yamada & O’Leary, 1977) and its mass spectrum showed the presence of 1.00 zyx f 0.02 deuterium atom. (R)-(-)-4-Phthalimid0[4-~H]butyric Acid. To 4.7 mL of water containing 283 mg of the deuterated 4-aminobutyric acid prepared above and 310 mg of sodium carbonate was added 730 mg of N-carboethoxyphthalimide and the mixture was stirred at 20 O C for 30 min. The solution was filtered and then acidified, and the precipitated (R)-( -)-4- phthalimid0[4-~H] butyric acid was filtered, washed with cold water, and dried, mp 114-1 15 O C (lit. 115-1 17 OC; Gabriel & Colman, 1908). Methyl (R)-( -)-4-Phthalimido[4- 2H] butyrate. Esteri- fication of the above compound with diazomethane in ether gave a quantitative yield of methyl (R)-(-)-4- phthalimid0[4-~H] butyrate, which was recrystallized from ether, mp 88-89 OC (lit. 89-90 OC; Gabriel & Colman, 1908). (S)-(-)-[2-2H]Glycine. Hydrogen exchange of the pro-S hydrogen of glycine (345 mg) was conducted in 23 mL of 0.01 M phosphate buffer in D20, pD 7.8, containing 1.5 mg of py- ridoxal-P, 3 mg of tetrahydrofolic acid, and 3.3 mg of serine transhydroxymethylase under nitrogen for 5 days at 20 “C. The enzyme was then removed by precipitation with trichlo- roacetic acid and centrifugation and the solution was applied to a column of Dowex 50W (1 00-200 mesh, H+ form, 1 X 35 0 1978 American Chemical Society