Ornithine Cyclase (Deaminating)

The conversion of L-ornithine to L-proline by ornithine cyclase involves the deamination of the a-amino group prior to cyclization. Therefore, Z-oxo-Saminopentanoic acid and Al-pyrroline-Z-carboxylic acid are likely intermediates in the conversion. The single mole of NAD+ bound tightly to the holoenzyme appears to play a catalytic role in the conversion since a transient absorption peak at 340 nm is observed upon addition of substrate to the cyclase.


Heavy
Nitrogen Experiment Synthesis of [6-*~N]Omithine- Ornithine was synthesized from a-aminoadipic acid and potassium [lSN]azide (96.4 atom '% enriched, Mallinckrodt Nuclear, St. Louis, MO.) in a Schmidt degradation reaction by a procedure adapted from two sources (5,6). *This investigation has been supported by Research Grant l-ROl-A110951.02 from the National Institutes of Health.
Journal Article 6177 from the Michigan Agricultural Experiment Station.
$ Graduate Fellow under Title IV, National Defense Education Act. This work was submitted in partial fullfillment of the requirements for the degree of Doctor of Philosophy.
The newly synthesized amino acid was purified on Dowex 50 (H+form columns). dried. and dissolved in distilled water. The identits of the'amino $cid synthesized was confirmed as ornithine from (a) paper ionophoresii in 0.125 N sodium acetate (pH 5.2), 0.5 N acetic acid CDH 2.6). and 0.2 M formic acid CDH 2.0): 6) descending chromaiograph;r on Whatman No. 3Mk paper' developed in butanol-acetic acid-water (12: 3:5); and (c) by ascending chromatography on thin layer silica gel developed with chloroform-methanol-15yo ammonium hydroxide (36:46:20). Assay of the product by the reduced ninhydrin method (7) indicated the yield was 250 pmol or 16.6yo. Samples of the synthesized product and authentic ornithine were examined in the combined gas chromatograph-mass spectrograph after preparing TMS derivatives of each.

Enzyme
Reactions Involving [6-16N]Substrate-Labeled [&15N]ornithine and unlabeled ornithine were converted to proline in reaction mixtures similar to the standard anaerobic, radiochemical assay (3), except that Cue was added to these reactions to inhibit proline reductase activity.
Each reaction mixture contained 15 ~1 each of 0.25 M Tris chloride buffer (pH 8.0), 0.1 M mercaptoethanol, 1 mM NAD+, and 66 mM CuC12. The source of the cyclase was ammonium sulfate-treated and -dialyzed extract from Clostridium sporogenes, which still contained some proline reductase activity.
The reaction mixtures contained 75 ~1 of the extract (specific activity, 0.09), and the reactions were started by adding 35 ~1 of either 0.2 M labeled or unlabeled substrate.
The reaction mixtures were incubated for 2 hours at 37" to allow for maximum conversion to take place. The protein was heat-inactivated.
A radiochemical assay run concurrently indicated that about 3.3 pmol of proline were formed by the enzyme.
Some of the NH3 liberated by the cyclase reaction was trapped in glutamate by the use of glutamate dehydrogenase.
The following reagents for this reaction were added to each of the reaction mixtur& after heating to stop the cyclase activity: 30 ~1 each of 1.5 M Tris chloride buffer CDH 8.2). 50 mM a-ketoalutarate, 20 mM ADP, and 40 mM NADH. 'The reaction was star&d by adhing 8.5 units (10 ~1) of glutamate dehydrogenase.
The reaction mixtures were incubated at 30" for 1 hour. The protein was precipitated by heating each tube to 80" for 5 min.
The residual ornithine and newly formed proline and glutamate were separated from other reaction components by Dowex 50 (H+-form) column chromatography.
The amino acids were dried in a rotary evaporator and then dissolved in a minimum volume of distilled water.

Analytical
Methods-Samples containing 100 pg each of 6-15Nlabeled or unlabeled ornithine and samples of the resuspended enzymatic products estimated to contain approximately 100 pg of proline and 35 rg of glutamate were dried at 50" in small (6 x 50 mm) tubes inside screw-capped tubes (13 x 100 mm). After drying, the sample tubes were maintained under dry nitrogen or argon. The dried samples were then solubilized in 50 ~1 of aceto-nitrile and 50 ~1 of bis-(trimethylsilyl)trifluoroacetamide containing 1% (v/v) trimethylchlorosilane (8,9). Each sample was heated to 77" for at least 30 min to facilitate the trimethylsilylation reaction.
Samples ( other prominent peaks obtained from the tetra-TMS-ornithine peak indicated the b-amino group was 28.0 atom y0 enriched for l5N. The method of synthesis ensured that all label was directed to the &position. TMS derivatives of the proline and glutamate from reaction mixtures were readily separated from each other and from residual ornithine and several other compounds present in the derivatized sample by gas chromatography (Fig. 1). Four ion peaks from the mass spectrum of proline were examined (Table I). The parent ion, (TiK%$-proline (M), was not included since it was eit.her of very low intensity or absent in every mass spectrum taken. The largest molecular ion consistently present was that at m/e = 244 (Fig. 2) which corresponds to 112 -15 (the loss of one methyl group).
Other prominent ions used were those at m/e = 216, 142, and 70. Each of these contain the nitrogen from the proline ring. The ion at m/e = 216 may be regarded as TM-N=CH---COO-TMS.
The base peak at m/e = 142 (U -117) represents one of the expected products of cx fission 0 Only a single value could be determined from these data. (15) : TMS-pyrroline. The final ion considered is that at m/e = 70 which may be regarded simply as the pyrroline ring from proline.
It is evident from a comparison of the mass spectra (Fig. 2, a and 6) and the ion ratio intensities (Table I) of proline produced from unlabeled and from [WN]ornithine that the 15N and thus the &NH3 group of ornithine is conserved in proline.
The calculated atom percentage of 15N in the proline formed (28.6 f 2.6) is the same as that determined for the &amino group of [WSN]ornithine used as the substrate for the cyclase reaction. Four ion peaks from the mass spectrum of glutamate were also examined (Table II and Fig. 3). A small but consistent parent ion of m/e = 363 was present which suggested a tri-TATS derivative. The readily identifiable second ion of m/e = 348 (~11 -15) results from the loss of one methyl group from the parent ion. The third useful ion was located at m/e = 246 (n!! -117) and corresponded to an (Y fission product: (T&%)2-(OOC-CH&H&EKH).
The last molecular ion examined was m/e = 218 (Ji -145). The ion was regarded as a /3 fission product containing both the amino group and the ac-carboxyl group (TMS-+NH=CHCOOTMS).
All four of these ions contained the NH3 group of glutamate which was generated from the 7465  ammonia liberated during the conversion of ornit'hine to proline by the cyclase. Comparisons of the mass spectra (Fig. 3, a and  b) and of the ion intensities (Table II) of glutamate from reaction mixtures in which unlabeled and [dJ"N]ornithine were substrates for the cyclase reactions indicate there was no significant enrichment of laN. Therefore, the a-NH3 group of glutamate must be derived from the a-NH3 group released from ornithine by the cyclase.
Role of Bound NAW-Following addition of substrate to highly purified cyclase a transient absorption peak at 340 nm appeared (Fig. 4). The data were corrected for absorbance due to both enzyme and NAD+.
Thus, the net absorbance shown was most likely due to a transient buildup of reduced NAD+ (NADH).
Addition of a second aliquot of substrate caused the reappearance of the absorbance peak at 340 nm. Calculations based on the cyclase containing 1 mol of NAD+ per mol of enzyme, the protein concentration (25.2 mM), the molar absorbance for NADH (6.2 x 1Oa NT' cm-l), and the peak absorbance value (0.039) indicate that under these conditions 24.8% of the bound NAD+ may be present in the reduced state.
Incorporation of NA DT into Proline and Oxidation of NA DH-There was no apparent incorporat.ion of tritium from NAD'i TIME ( Cyclase (29.5 pg, specific activity, 0.86) added to each reaction tube contained 0.5 Nmol of ornithine and 2.5 nmol of NAD+.
In a control reaction, 0.24 rmol of proline was formed when 2. into proline during the cyclase reaction (Table III). It also appeared that the enzyme failed to incorporate tritium from NADT into proline when A'-pyrroline-2carboxylic acid was added.
Incorporation of very low percentages of tritium from either the A form or the I3 form of NADT could have occurred without detection in this experiment.
An attempt was made to demonstrate the Al-pyrroline-5carboxylic acid-dependent oxidation of NADH using purified enzyme which had been treated to resolve NAD+ as described in the companion paper (4). This enzyme preparation had a specific activity of 0.33 unit per mg and the conversion of L-[U-Wlornithine to n-[U-Wlproline was stimulated 2.8 times by the addition of 0.1 mM NAD+ to reaction mixtures.
Reaction mixtures of 0.5 ml total volume contained 25 mM Tris'HCl (pH 8.0), 1 mM dithiothreitol, 0.2 mM NADH, approximately 40 mM Ai-pyrroline-2-carboxylic acid, and 80 pg of enzyme. All reaction components except enzyme were equilibrated at 40" in a Gilford model 2000 spectrophotometer, and after addition of enzyme, the absorbance at 340 nm was monitored for 15 min. No change in absorbance was observed. If the half reaction had proceeded at the same rate as the conversion of ornithine to proline, there should have been a AAa4a of about -0.32 per min. Previous experiments (2) with extracts of Clostridium botulinurn which also contained cyclase also failed to demonstrate NADH oxidation in the presence of A'-pyrroline-2-carboxylic acid. Therefore, if this compound is a true intermediate in the cyclase reaction and if NADH is used for its reduction, one or both of these compounds is apparently irreversibly bound during t.he reaction.

DISCUSSION
Previous data (2,3) indicated that the conversion of ornithine to proline by ornithine cyclase might proceed in one of two possible directions.
The direction taken would obviously depend on which ammo group was involved in the deamination.
Loss of the b-amino group would lead to the formation of glutamic y-semialdehyde which would undergo ring closure to form Alpyrroline-5-carboxylic acid. Deamination in the ol-position would form 2-oxo-5-aminopentanoic acid and Ai-pyrroline-2. carboxylic acid. The conservation of 15N from [6-15N]ornithine in proline during the conversion of ornithine to proline by the cyclase enzyme established with certainty that it is the a-amino group of ornithine which is removed.
The appearance of a transient peak at 340 nm after addition of substrate and the presence of 1 mol of tightly bound NAD+ per mol of cyclase (4) strongly suggest that the absorbance is due to enzyme-bound NADH. Thus, the enzyme-bound NAD+ appears to function as a catalytic cofactor in an enzyme-bound oxidative deamination and reduction as postulated.
The failure of NADT to exchange onto the cyclase is not unreasonable since the affinity for NADC is very high (apparent K, = 6.1 PM, see Reference 3) and NADH generally binds more strongly to a dehydrogenase than NAD+ (16). Of course, it is possible that isotope selection might hinder the exchange, since Peville et al. (17) noted a large negative isotope effect with UDP-Dglucose 4'-epimerase using UDPn-glucose-4-T as substrate. In an earlier paper (3), Costilow and Laycock showed that no tritium was incorporated into nonexchangeable positions in proline when the cyclase reaction was run in an assay mixture containing TzO. Again, barring complete isotope selection, the lack of tritium incorporation into proline is consistent with an enzyme-bound oxidative deamination-reduction mechanism involving NAD+, since all known dehydrogenases add and remove hydrogens from nonexchangeable positions, while the other involved hydrogen is in a freely exchangeable position on the substrate molecule (18). Also the UDP-n-glucose 4'-epimerase has a postulated internal oxidation-reduction mechanism and fails to incorporate tritium from TtO into the product (19).
Thus, we propose that the conversion of L-ornithine to Lproline by ornithine cyclase (deaminating) proceeds as follows: (a) enzyme-bound ornithine is oxidized to 2-oxo-5-aminopentanoic acid with the reduction of enzyme-bound NAD+ and the release of NH,, (5) the bound 2-oxo&aminopentanoic acid undergoes ring closure to form Al-pyrroline-2-carboxylic acid, and (c) the pyrroline ring is then reduced to form L-proline using the bound NADH as the reductant.
The proline is then released from the enzyme.
However, attempts to demonstrate the half reaction between NADH and Ai-pyrroline-5-carboxylic acid were unsuccessful. Therefore, if these two compounds are true intermediates in the conversion of ornithine to proline, they are apparently irreversibly bound to the enzyme during the reaction.
Acknowledgments-We are greatly indebted to C. C. Sweeley, Department of Biochemistry, Michigan State University, for the use of the combined gas chromatograph-mass spectrograph. Our thanks also to Jack Harten and Norman Young for excellent technical assistance.