Decarboxylation mechanism of the n-terminal glutamyl moiety in γ-glutamic acid and methionine containing peptides

~

Pergamon

Radiat. Phys. Chem. Vol.47, No. 3, pp. 507-510, 1996 Copyright © 1996ElsevierScienceLtd 0969-806X(95)00137-9 Printed in Great Britain.All rights reserved 0969-806X/96 $15.00+ 0.00

DECARBOXYLATION MECHANISM OF THE N-TERMINAL GLUTAMYL MOIETY IN 7-GLUTAMIC ACID AND METHIONINE CONTAINING PEPTIDES KRZYSZTOF BOBROWSKI 1'2 and CHRISTIAN SCHONEICH 3 qnstitute of Nuclear Chemistry and Technology, 03-195 Warsaw and Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-252 Warsaw, Poland, 2Radiation Laboratory, University of Notre Dame, Notre Dame, IN 46556, U.S.A. and 3Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66045, U.S.A. Abstract--The reaction of hydroxyl radicals with F-glutamyl-methionine and F-glutamyl-glycyl-methionyl-glycine at neutral pH results in similar N-terminal decarboxylation efficiency. The underlying mechanism involves an intramolecular proton transfer from the protonated N-terminal amino group of the glutamyl moiety to an initially formed hydroxy sulphuranyl radical at the methionine residue. This process leads to the formation of a three-electron bonded [ > S.'. NH2]+-peptide intermediate subsequently decomposing into CO2 and an ~t-amino radical of the structure H2N-CH'A2H2~H2-C(~-----O)-NHpeptide. This radical has been identified via reduction of a moderately good electron acceptor such as p-nitroacetophenone (PNAP). The arrangement within a sterically favourable 5-membered ring, as observed with methionine, is not a necessary prerequisite for the formation of [>S.'.NH2]+-type intermediate. Mechanistically, the formation of CO2 and an a-amino radical suggests the occurrence of an intramolecular electron transfer from the carboxylate group to the electron-deficient center at the nitrogen within the S.'.N-bond followed by homolytic bond breakage of the carbon-carboxylate bond. The decarboxylation benefits in particular from stabilization of the arising carbon-centered radical by a free lone pair from the or-amino group. This process seems to occur well in larger peptide structures provided they contain an N-terminal carboxyl group ct to an amino function and they are flexibleenough to allow the protonated amino function to interact with the hydroxyl sulphuranyl radical at the methionine residue.

INTRODUCTION The reaction of hydroxyl radicals with methionine proceeds via an initial formation of a hydroxyl sulphuranyl radical [reaction(l)]: HO" + Met(S) ---, Met(S'-OH)

(1)

The interaction of such hydroxy sulphuranyl radical with other functional groups present in peptides (e.g. amino, hydroxy or carboxyl groups) might be of biological significance. In recent studies (Bobrowski et aL, 1991a) it has been shown that the reaction of the hydroxyl radical with the methionine model peptide ?-glutamyl-methionine (~:-Glu-Met) and S-alkyl glutathiones at slightly acidic to neutral pH leads to the elimination of CO2 from the N-terminal carboxylate group with parallel formation of or-amino type radicals. This process occurs even though sulphide function and N-terminal carboxyl group are separated by a peptide bond. In analogy to methionine (Hiller et al., 1981; Asmus et al., 1985), a direct interaction of the hydroxy sulfuranyl radical (>S'--OH) and an amino function located ct to a carboxyl group has been considered to be responsible for the decarboxylation despite that both reaction centres (sulphide and carboxyl function) are not

located within the same amino acid unit. Such a prerequisite has been, for example, found to be required for C-terminal decarboxylation pathway via electrontransfer from carboxylate to a sulfur-centred radical cation (Bobrowski et aL, 1991b). Moreover, it has been specifically noted (Bobrowski et al., 1991a) that in the ~:-Glu-Met and S-alkyl glutathione derivatives an intramolecular arrangement within a favourable 5membered ring, as observed with methionine, was not necessary for the N-terminal decarboxylation. In the present paper, this work is extended to the tetrapeptide ?-glutamyi-glycyl-methionyl-glycine (F-Glu-Gly-Met-Gly). This peptide contains both an N-terminal carboxyl group which derives "activation" from the or-amino group, and a C-terminal carboxyl group, separated from the methionine by one peptide bond. Insertion of another glycine residue results in an increase of the separation between 7-Glu and Met as compared to the peptide ~:-Glu-Met. It seems reasonable to expect a similar interaction between a hydroxy sulphuranyl radical at Met and the protonated amino function located at the ),-Glu residue provided that this peptide is flexible enough to allow this interaction to occur fast enough to compete against alternative processes such as formation of sulphur-centred radical cations.

507

Krzysztof Bobrowski and Christian Schtneich

508

Interaction between remote functional groups has been recently proposed (Grierson et al., 1992) to explain formation of ~-amino radicals in glutathione. This reaction occurs via intramolecular transfer of the hydrogen atom (attached to the ~-carbon of the glutamic acid moiety) to the thiyl radical site located on the cysteine residue within the conformation with close approach of the reaction centres. EXPERIMENTAL

Materials

The peptide ~,-Glu-Met was obtained from Bachem Bioscience Inc. (Philadelphia, PA) as the best available grade and was used without further purification. The peptide F-Glu-Gly-Met-Gly was synthesized by standard solid phase methods using the Fmoc protected amino acids. The peptide was characterized by FAB mass spectrometry and its purity checked by HPLC. The other chemicals were obtained as follows: p-nitroacetophenone (PNAP) and perchloric acid (HCIO4) were purchased from Aldrich; reagent grade NaOH was from T. J. Baker. Solutions

accelerator. A description of the pulse radiolysis setup and data collection system is reported elsewhere (Schuler, 1985; Janata and Schuler, 1982). The experiments were carried out with a continuous flow of the sample. Absorbed doses were in the order of 2-4 Gy (1 Gy = 1 J / k g ) corresponding to an average concentration of radicals of (1.22.5) x 10-tM for a radiation chemical yield of G = 6.0 (g denotes the number of species formed or converted per 100 eV absorbed energy; G = 1.0 corresponds to 0.1036 /~mol per J absorbed energy in aqueous solution; for practical purposes the G unit rather than the SI unit is used throughout this paper. RESULTS AND DISCUSSION

Based on the mechanism for the OH-induced oxidation of X-Met (Bobrowski et al., 1991b), 7-GluMet and of S-alkyl glutathiones (Bobrowski et al., 1991 a) the decarboxylation of the peptide ?-Glu-GlyMet-Gly may result in the formation of only one type of product radical, the ~-amino radical of the structure H 2N - C H ' - C H 2 - C H 2-C(~-----O)-NH-CH2

All solutions were made up freshly in water purified by a Millipore Milli-Q system. The pH of the solutions were adjusted by adding sodium hydroxide or perchloric acid. They were subsequently deoxygenated by bubbling high-purity nitrous oxide (N20) through them. Pulse radiolysis

All pulse radiolysis experiments were performed by applying 10 ns pulses of high-energy electrons from the Notre Dame 7 MeV ARCO LP-7 linear

60,000-

-C(~-----O)-NH-CH(CH2-CH2-S-CH3) ~(=-O)-NH-CH:--COO-

(1)

Formation of the H3N+~[~H(COO-)-CHE~H2 C(~'~-~-O)-NH-CH2- C(~-------O)-NH-CH(CH2-CH2-S-CH3)-C(=O)--NH-CH 2" radical is prohibited by the separation of the sulphide carrying Met and the C-terminal carboxylate of Gly (Bobrowski et al., 1991a,b). Such ~t-amino radicals can be probed by one-electron reduction of moderately good electron

b

40,00020,O00-

f

X

O60,0007

&

6

6

;2

1'6

6

6

1'2

1'6

a

°E 40,000X

20,000" OTime

~s

Fig. 1. Optical absorption at 360 nm (expressed in terms of G x e) as function of time in a pulse-irradiated, N20-saturated aqueous solution containing: (a) 10 -3 M ~-Glu-Gly-Met-Gly and 1.5 x 10 -4 M P N A P at pH 6.4: (b) 10-3 M T-Glu-Met and 1.5 x 10-4 M PNAP at pH 5.9.

Decarboxylation mechanism of glutamyl moiety acceptors such as p-nitroacetophenone (PNAP) (Hiller and Asmus, 1983; Bobrowski et al., 1991a) ( E ~ = - 0 . 3 5 8 V ) (Meisel and Neta, 1975). This approach was applied to detect the potential presence of the H2N-CH'-CH2-CH2-C(:=O)-NH-peptide radical (1) derived from the ?-Glu-Gly-Met-Gly peptide. H 2N - C H ' - C H 2 ~ C H 2 - C ( = O ) - N H -peptide + PNAP-~ H2 N +=CH-CH2-CH2 - C ( = O ) - N H - p e p t i d e + PNAP-"

(2)

Reaction (2) is best observed by monitoring the

formation of the PNAP-' radical anion absorbing at 360nm (E360= 17,600dm3mol -~ cm -~) (Adams and Wilson, 1973). Figure l(a) shows a trace of the optical absorption at 360 nm recorded as a function of time in an N20-saturated solution containing 10-3M 7-Glu-Gly-Met-Gly and 1.5 x 1 0 - ' M PNAP at pH 6.4. Formation of the 360 nm absorbanee was of first-order kinetics with the half-life for the PNAP-" formation being 1.8#s in this particular experiment corresponding to k2 = 2.6 x 109 M -~ s -j. This value agrees well with that measured for the reaction of ~-amino radicals derived from methionine (Hiller and Asmus, 1983). The yield of PNAP-" (G = 2.7) accounts for ca 45% of the initial OH radical yield. One electron reduction reaction similar to equation (2) was also used as a probe for the formation of =-amino radicals derived from ~,-Glu-Met. These

HO'+

509

experiments were generally performed in N:Osaturated solutions (pH 5.9) of 10 -3 M 7-Glu-Met and 1.5 x 10-4M PNAP. Inspection of the trace of the optical absorption at 360 nm as a function of time [Fig. l(b)] reveals that the formation of PNAP-" occurs with a similar rate as compared to the tetrapeptide. Moreover, the yield of PNAP-" of G = 2.9 [in a good agreement with previous measurements (Bobrowski et al., 1991a)] corresponds to 50% of the OH radicals indicating that N-terminal decarboxylation occurs with the comparable ef6ciencies in both peptides. Comparable yields of PNAP-" (G---2.3) were also obtained with S-methylglutathione at similar experimental conditions (pH 6.1), 10-3M of S-methylglutathione (Bobrowski et al., 1991a). All these results are in accord with the formation of a three-electron bonded [> S.'.NH2 +peptide intermediate, as observed with methionine (Hiller et al., 1981; Asmus et al., 1985), which subsequently undergoes electron-transfer and parallel decarboxylation. Contribution of the ~t-(alkylthio)alkyl radicals to the total yield of PNAP-" is excluded due to their weaker reducing properties. This was confirmed by the lack of PNAP-" (Bobrowski and Pogocki, unpublished results) and 4-CB (Bobrowski et al., 1992) formation for 2,2'-thiodiethanoic acid where decarboxylation leads exclusively to ~t-(alkylthio)alkyl radicals but not ~t-amino radicals. The results are rationalized by involving a mechanism, displayed in Scheme 1 (below).

7 Glu Gly M e t GIy

(3) ~.-~" 0 ~ 0 -OC-CH-CH~CHI~-NH-CHI~-NH-CH-~-NH-CH~CO 2NHa

CH 2

2

0 0 HC-C-NH-CH-C-NH-C

(4)

• /CH HO-S CH 3

"

0

2~ CH ,. 2

2

F-CH H~N+~ d ~*'"H20

o.

o01

oii

I'~C-C-N H-C H~C-NH-FH'O-NH-CH~CO ~ CH / 2

"O~ C ' C H

H~N H

XCHs

o

o

O

~C-6-NH-CH~C:-NH-/CH-C-NH-CH,~CO~ CH • C'H 2

C H=

H"~N

S

(8)

+

o

H N+ H

CH3

CO 2

Scheme I

2

. CH iS-CHs

~o.H L (5)

+

,'.

I - CH, S

~C~

oLT 6)

o

~C~"~:'NH'cni(~'NH'/CH'~;'NH'CI'~CO~ CH C / 2 "O~ -CH

CH

2

o o o . u H-/CH-C-NH-CI' gl l'.~C-C-NH-CH~C-N ~CO CH CH=

z 2 c'CH.~

"+S

H

H

"0

/CH,

H-,N

6

(7)

C H2

0 H-C- N H - C H - C O -

I C H2

CH 2 CH 2

S

4 CH 3

510

Krzysztof Bobrowski and Christian Sehgneich

The initial oxidation step in the overall mechanism of decarboxylation of N-terminal 7-glutamyl moiety is a formation of a hydroxy sulphuranyl radical at the methionine moiety (2) [reaction (3)]. This adduct can subsequently eliminate the hydroxide ion using protons provided intramolecularly by the protonated amino group [reactions (4) and (5)]. The hydroxy sulphuranyl radical (2) can also decompose via several additional routes: spontaneous and/or protoncatalyzed dissociation and displacement involving a second peptide molecule. The occurrence of these processes in competition to the proton-transfer route accounts for the observed yield of PNAP-" which is in all cases < 50% of the initial OH radical yield. The final elimination of water [reaction(5)] yields the short-lived [ > S.'.NH2] +-peptide intermediate (3). That type of intermediate has recently been identified by pulse radiolysis employing nanosecond time resolution in Ser-Met and Thr-Met peptides (Schgneich et al., 1994) and S-alkylglutathiones (Bobrowski et al., unpublished results). The intermediate 3 exists in equilibria with its acyclic derivatives 4 and 5 [reactions (6) and (7)]. Species 4 represents an amino acid N-centered radical cation, that can undergo an intramolecular electron transfer from the carboxylate group to the oxidized amino function followed by homolytic breakage of the carbon--carboxylate bond [reaction (8)]. The decarboxylation benefits in particular from stabilization of the arising carboncentered radical by a free lone pair from the ~-amino group (6). The present investigation on the decarboxylation of 7-Glu-Met and 7-Glu-Gly-Met-Gly peptides demonstrates that the hydroxy sulphuranyl radical at a methionine moiety can interact even with remote functional groups in peptides. This type of interaction can be monitored by model reactions such as the decarboxylation of N-terminal glutamyl moieties via intramolecular proton transfer. We are currently extending these type of studies to even larger flexible or constraint peptides in order to evaluate if our model reaction may permit the determination of absolute rate constants for the interactions of functional groups in peptides and proteins. Acknowledgements--This work was supported by the Office of Basic Energy Sciences of the DOE (K.B.) and by

the Association for International Cancer Research (AICR)(Ch.S.) This is Contribution No. NDRL-3756 from the Notre Dame Radiation Laboratory. REFERENCES

Adams G. E. and Wilson R. L. (1973) Ketyl radicals in aqueous solution. Pulse radiolysis study. J. Chem. Soc. Faraday Trans. 1 69, 719 Asmus K.-D., G6bl M., Hiller K.-O., Marling S. and M6nig J. (1985) S.'.N and S.'.O Three-electron-bonded radicals and radical cations in aqueous solutions. J. Chem. Soc. Perkin Trans. 2, 641.

Bobrowski K., Marciniak B. and Hug G. L. (1992) Carboxybenzophenone-sensitized photooxidation of sulfur-containing amino acids. Nanosecond laser flash photolysis and pulse radiolysis studies. J. Am. Chem. Soc. 114, 10279. Bobrowski K., Schgneich Ch., Holcman J. and Asmus K.-D. (1991a) 'OH Radical induced decarboxylation of y-glutamylmethionine and S-alkylglutathione derivatives: evidence for two different pathways involving C- and N-terminal decarboxylation. J. Chem. Soc. Perkin Trans. 2, 975. Bobrowski K., Sch6neieh Ch., Holcman J. and Asmus K.-D. (1991b) 'OH radical induced decarboxylation of methionine-containing peptides. Influence of peptide sequence and net charge. J. Chem. Soc. Perkin Trans. 2, 353. Grierson L., Hildenbrand K. and Bothe E. (1992) Intramolecular transformation reaction of the glutathione thiyl radical into a non-sulphur-centered radical: a pulseradiolysis and EPR study. Int. J. Radiat. Biol. 62, 265. Hiller K.-O. and Asmus K.-D. (1983) Formation and reduction reactions of a-amino radicals derived form methionine and its derivatives. J. Phys. Chem. 87, 3682. Hiller K.-O., Masloch B., Ggbl M. and Asmus K.-D. (1981) Mechanism of the OH radical induced oxidation of methionine in aqueous solution. J. Am. Chem. Soc. 103, 2734. Janata E. and Schuler R.H. (1982) Rate constant for scavenging e~qin N20 saturated solutions. J. Phys. Chem. 86, 2078. Meisel D. and Neta P. (1975) One-electron redox potentials of nitro compounds and radiosensitizers. Correlation with spin densities of their radical anions. J. Am. Chem. Soc. 97, 5198. Sch6neich Ch., Zhao F., Madden K. P. and Bobrowski K. (1994) Side chain fragmentation of N-terminal threonine or serine residue induced through intramolecular proton transfer to hydroxy sulfuranyl radical formed at neighbouring methionine in dipeptides. J. Am. Chem. Soc. 116, 4641. Schuler R. H. (1985) Computerized instrumentation in chemical experiments. Chem. Educ. (Ind.) 2, 34.