The catalytic decarboxylation of oxaloacetic acid by thermally prepared poly-α-amino acids

.\IKHIVES

OF

HIOCIIEMISTRY

The Catalytic

ANI)

BIOPHYSICS

Decarboxylation Prepared

Ecrobiology

118, -l6Gl74

Division,

AVLCS

(IMi)

of Oxaloacetic Poly-a-amino

Acid

by Thermally

Acids

lkwurch

Center, :VAS.-L, Moffelt

Received

Jrmr

Field,

Culifornia

24, 1%X

Thermally prepared poly-n-nmino acids c:~trllyzc the decnrboxylation of oxaloacetic acid to pyruvic acid. Such act.ion, at pll 5.0 nnd under conditions yielding pseudo first-order kinetic-s, is catnlyt.ic, rather th:m stoichiomctric~. Polymers contuining predominsntlg or solely residues of lysine nre 1he most. effective; some sre up t,o nbout 15 times mow ac-1ivc 1h:ui the eqilivnlent, amount of free lysinc. SIulLiple ssmpies of s particular type of tlwrmal polymer eshibit quite similar (&lO~o) levels of :icLivity. The action of the lysine-ric*h polymers is rather selective, 0s several other acids are not, decnrboxylatcd, Ilndcr the condit iotas routinely used with oxsloacctic acid. The possible prebiologicnl conditions of synt.hesis of the thcrmol polymers permit inferences in the context of biochemic~al origins.

The preparation of polynrlhydro-cY-~trllillo acids by the process of heating the proper proportion of dry monomers has been described (l--G). The product:: of the thermal condensations can rungc in wmpositionul complexity from homopolymcrs, c.g., polynspartic acid (2)) polylysinc (3)) t,o heteropolymers that wnttin somc~ proportion of each of the 18 cwmmon amino acids (4, 5). The latter thermal polymers txhihit many prop&k in wmmori with prolcin in xtldition t,o cwitcnt. of amino acids (1, 4, A), and they haw hccn tcrmetl pwteinoids (4). 13ccausc they arc prepared under prcsumc~d prcbiological conditions, proteinoids have been suggested as models of abiotic protein (1, 5, 6). Orie property of protcinoids cwrently under investigation is that, of c:lt.alytic activity. ‘l’hc first, reports of c:l.tiLlyt,ic action by protcinoids were for the hydrolysis of p-nitrophcnyl :wc:t.:~te (5, 7-9). I’rotcinoids cotlt:Litling histidinc were, in some cases, mow t.h:in 10 times as effective in hyI Sat ionnl Acndcmy of Sciences Post doctoral I~rsc:trch Ass0ciat.e. Present address: I)epurt.ment ot’ Biology, RIassachusetts Inst,itllte of Technolfogy, (::lmhridge, I\~l:lss:l~hclsetts.

drolyzing this unnatural and labile subst,rat.e than was the equivalent amount of histidine. Such preparations were almost wmpletely inactivated by heating in buffered solution. The simultaneous presence of residues of histidine and of the cvclic imide form of aspartic acid was rcqiired for optimal activity. Other laboratories have investigated the action of thcrmnlly prepared polyamino acids on this substrate (10, II), including studies of inhibition by inhibitors of choline organoy)hosphonls cskrase (1 1). SubscqucntJy, t,hcrm:tl polyamino :wids have been shown to promote or :welerate conversions of mtturally ocsubstxates. t’roteinoids promote curring the formation, nuti subsequent decarboxylation, of glucuronic: acid from glucose (12) ; pyruvic acid is dcc~arbosylatetl in the presence of the thermal polymers Lo give primarily acetic: acid (13, 14). Zinc salts of proteinoids rich in dicarboxylic amino acids (14), and zilic*-c:olit.uining microspheres made from prot.einoids (15), promote the hydrolysis of ATI’; tho action of the miwosphcrcs is reported to be 11or1catalytic (15). The above reactions with natural substrates have frequcnt~ly used

4G8

CATALYSIS

BY THERMAL

quite high ratios of polymer to substrate, and after l-3 days of action, have yielded only a fraction of the theoretical amount of product. It was therefore thought desirable to demonstrate a more rapid proteinoidaccelerated reaction, using a naturally occurring substrat.e under conditions that would allow one to determine whether the reaction were truly catalytic, or merely stoichiometric. The proteinoid-accelerated decarboxylation of oxaloacetic acid (OAA) was investigated in this context. A preliminary report of this investigation has appeared (16). MATERIALS

AND

METHODS

Proteinoids rich in dicarboxylic amino acids were prepared after the method of Fox and Harada (4)) and were employed as the sodium salts. Thermal polylysine was prepared at 195” C by the method of Harada (3). Lysine proteinoids (5) were prepared at 195” C from reaction mixtures composed of 12 parts by weight of L-lysine free base, 10 parts of an equimolar mixture of 16 basic and neutral L-amino acids (4) and 1 part each of Laspartic acid and L-glutamic acid. The latter two types of polymers were employed as the HCl or HOAc salts. All preparations were dialyzed against running distilled water for 3 days. Any trace amounts of insoluble materials were removed prior to lyophilization; subseqrlent experiments were thus conducted on completely soluble materials. Some polymers were prepared and processed under aseptic conditions (cf. 12). After aseptic dialysis, aliquots of the solutions were plated in quadruplicat,e on agar plates of (1) peptone, beef extract., dextrose; (2) peptone, yeast extract, dextrose; and (3) malt extract. After two-weeks’ incubation at, ambient temperature, only an occasional colony was observed; most plates were devoid of colonies. Some polymers were analyzed for content of met,al ions by the Morse Laboratories, Inc., Sacramento, California. The analyses indicated the presence of quite small amounts of Ba, Mg, Fe, Ca, Cu, and Na (totaling about 0.1 pmole/mg of polymer), possibly attributable to handling during analysis (17). A-grade L-amino acids and glycine were obtained from California Biochemical Corp. Los Angeles, California; L-lysine free base was from K and K Laboratories, Plainview, New York (lot no. 55793). Oxaloacetic, pyruvic, a-ketoglut.aric, malic, and malonic acids were California Biochemical Corporation products. Oxalic acid and potassium glucuronate were from Matheson, Cole-

POLYAMINO

469

ACIDS

man and Bell, Los Angeles, California. p-Glucose was from Nutritional Biochemicals Corporation, Cleveland, Ohio. Samples of polylysine prepared via the c-carbobenzoxy Leuchs anhydride were obtained from Mann Research Laboratories, Inc., New York, New York (lot M1804, molecular weight 75,000) and from New England Nuclear Corp., Boston, Massachusetts (lot LY4S, molecular weight 40,000). Lithium acetoacetate was prepared by the method of Hall (18). The decarboxylation of oxaloacetic acid (OAA) was followed manometrically at 30”, using a slight modification of the procedure of Bessman and Layne, Jr. (19). A typical reaction mixture contained l&4.0 mg of thermal polymer and approximately 10 pmoles of freshly dissolved OAA, in a total volume of 2.1 ml of 0.2-M acetate buffer, pH 5.0. The polymer-catalyzed reactions were usually followed to cu. SO-SO% completion, which required 1-2 hours or less. Control flasks containing 0.03 M NiC12 were used to determine the total amount of OAA initially present (cf. 19) in each experiment, this value then being used in the construction of first-order plots for the polymer-catalyzed reactions. First-order rate constants obtained in this manner were in close agreement with those obtained in experiments in which the reactions were followed to greater than 99% completion, or by the method of Guggenheim (20) ; these two methods were less convenient. In all experiments, controls for spontaneous decarboxylation were utilized. The spontaneous rate (0.003 per minute) was subtracted from the rates observed in the presence of polymer. The corrected values thus obtained are termed K’, and are converted, where necessary, to unit concentration of polymer (1.0 mg/ml). A linear dependency of first-order rate constant upon the concentration of polymer employed, over a IO-fold range encompassing those routinely used, justified this latter treatment. (The corrected rat.e constants are actually apparent second-order rate constants, with units of liter per gram per minute.) Substrates other than OAA were t.est.ed in a similar manner. RESULTS

AND

DISCUSSION

First-order plots depicting the decarboxylation of OAA are shown in Fig. 1. The reactions, in the presence of 1.0 mg/ml of lysine-rich polymers, go to 80-90% completion in ca. l-2 hours. (Lysine proteinoid was usually employed at a concentration of 2.0 mg/ml, so that its action was nearly complete in 1 hour or less.) The speed of these reactions may be compared

ROHLFING TABLE ACTIVITIES

ON OAA

Material

IO 0

I 20

I 40

I 60 TIME,

I 00

I 100

120

min

FIG. 1. First-order plots depicting the decarhoxylation of OAA. Bot,h polymers were employed at 1.0 mg/ml in 0.2-N acetate buffer, pH 5.0. The initial OAA concentration was ca. 5 X 1W M. Reaction followed manometrically.

with those reported for the decarboxylation of pyruvic acid (13) and of glucuronic acid [with glucose being the initial substrate (12)], where much larger ratios of proteinoid to substrate caused the liberation of only a small percentage of the theoretical amount of CO2 in 24 or 72 hours. In reactions that were allowed to go to completion, each mole of OAA liberated one mole of COZ, identified as such in experiments that employed KOH in the Warburg flasks. Pyruvic acid was identified as a second product by thin-layer chromatography in three solvent systems (21) and by the melting point (219-221” C) and mixed melting point (219.5-221.5“ C) of the 2,4-dinitrophenyl hydrazone. The derivative of the authentic pyruvic acid melted ?.t 220-221” C [lit., 221’ C (22)]. -4 20-fold range of OAA concentration did not significantly alter the first-order rate constants observed with 0.2 mg/ml of polymer. III Table I are reported first-order rate constants for several samples of thermally prepared polyamino acids. The rate constants are corrected for the spontaneous rate, and are converted to unit concentration (1.0 mg/ml) of polymer. Acid-type proteinoids, which have been effective on

Acid proteinoids D A-1~ A-2 A-3 A-4 A-5 Lysine proteinoids F A (aseptic)d A (nonaseptic)” C-l c-2 c-3 c-4 Polylysine F A (aseptic)4 C-l c-2 c-3 c-4 (Spontaneous

I

OF THERMAL ACIDS~ K’,

min-1 X 103

1.3f0.36 0.5 0.3 0.4 0.3 0.6

POLYAMINO Average,

min-1 X 103

0.6f0.4*

11.9f1.5 11.4*0.3 11.4Ztl.7 11.5AO.5 11.5fl.O 12.2f1.3 ll.O~tl.6

12f0.4

49323 54f5 62f6 54f3 52f6 55f5 3.0f0.7)

54f4.0

a Assayed at pH 5.0 as described in the Methods section. Activity values are corrected for the rate of spontaneous decarboxylation and are converted to unit concentration of polymer (1.0 mg/ml). * Standard deviation. c Some of the polymers of this series contained less than the typical (6) proportion of aspartic acid. Activities were determined only twice, and standard deviations were not determined. d Prepared and processed under aseptic conditions and found to be devoid of microorganisms on three types of culture media. e Dialyzed without taking aseptic precautions.

other substrates (5, 12-15), exhibit very little activity on OAA. Because of their low level of activity, they were not investigated further. Lysine proteinoids are about 20 times more effective than the same weight of acid proteinoid [an observation that parallels results observed when glucose was used as the substrate (12)], and samples of polylysine are some 90 times as active. (On a lyeine-residue basis, polylysine was about 3 times as active as lysine proteinoid.) The reproducibility of assay

CATALYSIS

BY THERMAL

of a single sample of lysine-rich polymer, evaluated from 4 to as many as 20 times, is about &lo 70. Multiple samples of a particular kind of thermal polymer show quite similar levels of activity, illustrating that the degree of act’ivity is reproducible by synthesis ( f 10 %). This observation is in contrast to those made in other studies (5, 14). Polymers prepared and processed under aseptic conditions were as active as those for which no aseptic precautions were taken; thus the observed activity is not attributable to microbial contaminations. This conclusion was further indicated by the fact that no loss of activity was observed when buffered solutions of polymer were heated in sealed tubes in a boilingwater bath for 20 minutes. The rate of spontaneous decarboxylation is somewhat lower than the value of 0.006 per minute obtained under different conditions by Bessman and Layne, Jr. ( 19). The data in Table II are part of the evidence indicating that the reactions are truly catalytic, and not stoichiometric. Three successive aliquots of 10.0 pmoles of OAA were reacted with one aliquot of thermal polymer, the second and third portions of OAA being added after the preceding one had completely reacted. With each aliquot of substrate, the polymer/OAA ratio was initially near unity (weight or molar basis, expressing the molar concentration of polymer in terms of lysine residues). Essentially identical rates are observed with all three aliquots of substrate, both with polylysine and with lysine proteinoid. At the ratio of polymer to substrate employed, a progressive decrease in rate with successive charges of substrate would be predicted for a stoichiometric reaction (23).2 Addi2 It should be pointed out that this technique will not distinguish bet)ween a catalytic and stoichiometric reaction if the polymer/substrate ratio is quite large (e.g., 100/l), because only a small fraction of the reactive groups (e.g., 1%) in the polymer would be altered in a st,oichiometric reaction. Stoichiometric action with a second aliquot of substrate would thus proreed nearly as fast (e.g., 99%) as with the first aliquot. The maximum usable ratio of polymer/substrate is therefore dictated by the precision of the assay.

POLYAMINO

471

ACIDS TABLE

EFFECT ON RATE

II

OF SUCCESSIVE ALIQUOTS OAA5 K’. min-1

Catalyst

Polylysine (Sample

x 103

F)

Lysine proteinoid (Sample F)

1st 2nd 3rd 1st 2nd 3rd

65, 58, 57, 24, 25, 22,

60 54 53 24 25 21

OF

AVerage, min-1 X 103

(*7

58f4.2b rel. %)

23.5f1.7” (f7 rel. %)

a The 2nd and 3rd aliquots of OAA were added to the assay solutions after no further CO2 was liberated from the preceding one. With each aliquot of 10 pmoles of OAA, the calculated molar ratio of substrate to polymer (lysine residue basis) was unity. Respective weights of polylysine and lysine proteinoid were 1.2 and 2.4 mg in 2.1 ml acetate buffer, pH 5.0; 30”. Rate constants are not converted to unit concentration of polymer, but are corrected for slight (cu. 5%) dilution resulting from addition of 2nd and 3rd aliquots of substrate. The entire experiment required slightly over 6 hours. b Standard deviation.

tionally, with near equal amounts of polymer and substrate, nonlinear first-order plots would be predicted for a stoichiometric reaction (24-26) ; such plots, however, were linear for 80-90% of the reaction (Fig. 1). Finally, in the experiment depicted in Table II for polylysine, at least three equivalents of CO2 were liberated per equivalent of lysine residue present in the polymer, a fact further consistent with catalysis. The action of acid proteinoids on p-nitrophenyl acetate has also been shown to be catalytic (8). Various metal ions catalyze the decarboxylation of OAA (19) ; however, the activity of the thermal polymers was not due to possible metal ion contaminants. Analyses of samples of lysine proteinoid and of polylysine indicated very low levels of metals (ca. 0.1 pmole/mg), possibly attributable to handling during analysis (17). Synthetic mixtures of metal ions reflecting the analyses were prepared and assayed for catalytic activity in conjunction with the “equivalent” amount of polymer. The respective rates (X 103) observed for

472

ROHLFING TABLE ACTIVITIES

III

OF VARIOUS

CATALYSTS K’. k-1

Catalyst=

Dialyzed product Reactants Hydrolyzate of dialyzed productc Crude producta Acetylated dialyzed product”

X 10s

Polylysine Pr!ztiEid 6amp’e F, (Sample F)

506 3, 3 4, 5

116 2.5, 2.5 3.5,4.0

44, 45 1, 2

7, 7 1, 1

GMaterials assayed on an equal weight basis, except as noted in footnote c below. Conditions of assay were as described in the Methods section. Activity values are converted to unit concentration of polymer (1 me/ml). * Average of 4 determinations; duplicate analyses reported for the other materials. c Samples of the polymers were hydrolyzed (constant boiling HCl, evacuated, sealed tubes, 108” C, 48 hr), dried, taken up in water and redried twice, and were dissolved in acetate buffer to give a concentration equivalent on an amino acid residue basis to that of the parent polymer. d Received no dialysis. c Different parent polymers were used for acetylation; however, the activities of the parent polymers were quite similar to those reported here for “Dialyzed product.”

polymer (1.0 mg/ml), complement of metal ions, and admixture of the two were 48.5, 1.1, and 49.0 per minute for polylysine, and 11.7, 0.5, and 11.6 per minute for lysine proteinoid. Thus the polymers are far more active than are their complement of metal ions, and adding the latter to the polymer does not significantly affect the rate of reaction. Furthermore, treatment of the polymers on either Sephadex G-25 or Bio-Gel P-60 columns, which should remove metal ions, did not alter the level of activity. The possibility that the slight activity of the acid proteinoids might be due to contaminating metal ions was not ruled out. The proteinoid-promoted evolution of CO2 from glucose is stimulated by ATP or by Mg++ (12) ; however, 2 X 10-3-~ Mg++, and also ~O+M Mn++ [which, lie Mg++, can serve as a cofactor for the natural enzyme catalyzing the decarboxylation of

OAA (27)], had no stimulatory effect in the present system. The effect of ATP could not be evaluated, as admixture of 5 X ~O"-M ATP and lysine-rich polymers resulted in precipitation. The pH-activity profile for the lysine-rich polymers, over the range from 2 to 6, showed optimum activity near pH 5 or 6, and dropped with decrease in pH. The values observed at pH 3 were cu. $
CATALYSIS

BY THERMAL

However, possible structural differences, for example, t-peptide linkages (as), may be a contributing factor. The activity of the thermal polymers cannot be precisely compared with those reported for the nat’ural enzyme, because of differences in assay conditions and uncertain degrees of purity of the enzyme. The enzyme is undoubtedly hundreds or thousands of times more active than the thermal polymers. For example, values of 30, 900 (2i), and 70 (29) pmoles of CO2 produced per minute per milligram of protein have been reported for oxaloacetate carboxylases. The largest ratio of OAA/polymer tested (20 pmoles OAA per 0.4 mg of polymer) gave a value, calculated from progress curves, of 0.3 pmoles COZ per minute per milligram of polylysine. Energies of activation, determined over the temperature range of 20-40°, were 23, IS, 16, and 15 kcal, respectively, for spontaneous decarboxylation, lysine hydrochloride, thermal polylysine, and lysine proteinoid. Gelles (30) has reported a value of 23 kcal for the spontaneous decarboxylation of OAA anion; 6.4 kcal is reported for wheat-germ OAA carboxylase (27). The action of the thermal lysine-rich polymers is rather selective. No evolution of CO% was detected manometlically, under conditions in which OAA was 90 ‘i’o decarboxylated, when pyruvic, malic, malonic, or-ketoglutaric, glucuronic, oxalic, or aspart’ic acids, or glucose, were employed as substrates. Although other laboratories have reported (12-14) that thermal polyamino acids accelerate the decal boxylation of some of these substrates, the present findings do not refute such reports, because grossly different assay conditions have been used. (For example, other studies have employed much larger ratios of polymer/ substrate, different pH values, longer reaction times, and much more sensitive assay procedures.) The lysine-rich polymers did slowly decarboxylate acetoacetic acid, about $& as fast as OAA. Gelles (30) found acetoacetate anion to be spontaneously decarboxylated $50 as fast as OAA anion. p-Nitrophenyl acetate was hydrolyzed by the lysine-rich polymers. The

POLYAMINO

ACIDS

473

selective action of the polymers is perhaps a reflection of the relative stability of the substrates investigated. Other investigations (5, 6, 8) have shown that the amino acid composition of thermal polyamino acids, can be easily and controllably varied; the contribution to catnlytic activity of a particular kind of amino acid residue can therefore be evaluated (5, 8, 10, 12, 14). This fact has led to suggestions (S, 14) of the use of thermal polyamino acids as model compounds for investigations of the nature of the active sites of enzymes. The results of the present study are consistent with these suggestions. Thermal poly-a-amino acids are prepared under presumed prebiological conditions (1). The results of this investigation have shown the action of the thermal polymers 011 a naturally occurring substrate to he rapid, quantitative, selective, and, most importantly, truly catalytic. These findings thus augment other results (5, 8, 12-15) in suggesting that the action of heat on dry amino acids may be one way by which protein-like macromolecules with catalytic properties could have been formed abiogenically. KEFEl1ISNCES 1. Fox, 2.

3. 4. 5.

6.

7. 8. 9.

10.

S. W., iVakure 206, 328 (1965). VEDOTSKY, A., HARADA, K., AND Fox, S. W., J. Am. Chem. Sot. 80, 3361 (1958). HARADA, K., Bull. Chem. Sot. Japan 32, 1008 (1959). Fox, s. W., AND HARAD.4, K., J. Am. Chew Sot. 82, 3745 (1960). Fox, S. W., HARADA, Ii., AND ROHLFING, D. L., in “Polyamino Acids, Polypeptides, and Proteins,” (M. Stahmann, Ed.), p. 47. University of Wisconsin Press, Madison (1962). Fox, S. W., HARADA, Ii., WOODS, K. It., AND WINDSOR, C. It., AnA. Hiochem. Biophys. 102, 439 (1963). ROHLFING, 11. L., AND Fox, S. W., Arch. Biochem. Biophys. 118, 127 (1967). ROHLFING, 11. L., .~ND Fox, S. W., llrch. Biochem. i3iophy.s. 118, 122 (1967). Fox, S. W., AND ROHLFING, D. L., Abstracts of Papers Presented at the 141st Meeting of the American Chemical Society, Washington, I$. C., March 1962, Paper 11-C. NOGUCHI, .J., AND SATTO, T., in Ref. 5, page 313.

474

ROHLFING

11. USDIN, V. R., MITZ, M. A., AND KILLOS, P. J., Abstracts of Papers Presented at the 150th Meeting of the American Chemical Society, Atlantic City, New Jersey, September, 1965. Paper 43 C. 12. Fox, S. W., AND KRAMPITZ, G., Nature 203, 1362 (1964). 13. KRAMPITZ, G., AND HARDEBECK, H., Nuturwiss. 63, 81 (1966). 14. DURANT, D. H., AND Fox, S. W., Federation hoc. 26, 342 (1966). 15. Fox, S. W., “The Origins of Prebiological Systems and of Their Molecular Matrices,” p. 361. Academic Press, New York (1965). 16. ROHLFING, D. L., Federation Proc. 26, 524 (1966). 17. MORSE, G. H. (personal communication). 18. HALL, L. M., Anal. Biochem. 3, 75 (1962). 19. BESSMAN, S. P., AND LAYNE, E. C. JR., Arch. Biochem. Biophys. 26, 25 (1950). 20. GUGGENHEIM, E. A., Phil. Msg. 2, 538 (1926).

and Electro21. SMITH, I., “Chromatographic phoretic Techniques,” Vol. 1, pp. 263-264. W. Heinemann Medical Books, London (1960). 22. SUBRAMANIAN, I’., STENT, H. B., AND WALKER, T. K., J. Chem. Sot. 1929, 2490. 23. KOLTUN, W. L., NG, L., AND GURD, F. R. N., J. Biol. Chem. 238, 1367 (1963). 24. KOPPLE, K. D., AND NITECKI, D. E., J. Am. Chem. Sot. 83, 4103 (1961). 25. ADLER, A. J., FASMAN, G. D., AND BLOUT, E. R., J. Am. Chem. Sot. 86, 90 (1963). 26. CRUICKSHANK, P., AND SHEEHAN, J. C., J. Am. Chem. Sot. 86, 2070 (1964). Handbook,” pp. 27. LONG, C., “Biochemists’ 449-450. Van Nostrand, New York (1961). 28. HARADA, K., AND Fox, S. W., Arch. Biochem. Biophys. 109, 49 (1965). 29. HORTON, 9. A., AND KORNBERG, H. L., Biothem. Biophys. Acta 89, 381 (1964). 30. GELLES, E., J. Chem. Sot. 1966, 4736.