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On the non-enzymic transamination of γ-aminobutyric acid
ARCHIVES
OF
RIOCHEMISTRY
.4KD
BIOPHYSICS
On the Non-Enzymic
96, 650-652 (1962)
Transamination
c. S. ROSSI, F. OLIVO,
A. R,4BBSSISI
of r-Aminobutyric ANL)
Acid’
r\r. SII,IPR,4NDI
Investigation of chemical trnnsaminntion between pgridoxal or pyridoxnl phorIhate and the y-nminobutyric acid has been carried out,. It has thus been possible to give evidence for imine formation and for trnnsnmination betwew pyridoxal or pvridoxal phosphate and y-aminobutyric acid. Pyridoxal, iminc, and pyridoxamine have been separated by paper chromatography. Absorption spectra and behavior in acid and alkaline solutions are consistent with the hydrogen-bonded imine structure INTROD1-CTION
free phenolir group is so placed as to stabilizc the imiw through hydrogen bonding or, in tlic prescnw of appropriate metal ions, through the formation of chelatc rings:
Enzymic and non-enzymic reactions catalyzed by PLP’ are generally interpreted to occur through int’eraction between the formyl group of PL and the u-amino acid amino group with the formation of an imine : H 0 \
C-L
-0 /
II”
H I
\
CH I CHgOH II
HzC I
Bccording to Snell (1 j , in these imines a conjugate system of double bonds, extending from the a-carbon of the amino acid to the electronegative group, results in reduced electron density around t,he a-carbon atom, t’hus weakening the bonds to the carbon carrying the NH, group. In these imines, the ‘This work was supported by grants from Rockefeller Foundation and from Consiglio Sazionale dellr RicerchP. ’ A1bhrcviations used : 1’12, pyridoxal l~hor])hat~; PI,, pyridoxal ; PM, p~ridoxamin~~; C.4H.1. T-aminobutj+c avid : UV. ultraviolet 650
“Alanine-pyridoxal-Al+ + +” and “pyrurat’e-pyridoxamine-Al+ + +” imines have been recently isolated and characterized by Fasella et 01. (2). Participation of these int,ermediates in the transaminat,ion reactions was proved by the same authors. Since up to now chiefly a-amino acids haoc been considered in such “model syst~ellls,” it’ seemed interesting to investigate the possibility, extent, and mechanism of non-cnzymic transamination between PI, and a non-a-amino acid, such as GABA. In the cast of GABA, t’he amino groul) is so placed as to suggest a mechanism differcnt from thr formation of a chclate indicated as II, in which there is a bond bctwwn tlrr carboxy group and metal ion.
Moreover, the choice of this amino acid has been suggested by the particular role of GABA in cerebral metabolism in connection with PLP enzymes. MATERIALS
.\KD
METHODS
Itraction bctwwn PI, or PLI’ (2 X 10-l .l/) and GAB*1 (5 X 10-l .II) wcrc c:uri(yl out at 37°C. for dif%“crcnl t imrs. Appropriate I)uffcrs t~mployctl for the various pH’s wwc as follow: 0.05 :I! xclate at pH 4; pH 7-8, 0.05 JI phoq~hate: pH 12, 0.05 ill sodium l~ydrosidr. Altrmntivcly, nn rthanol-0.1 dl phosphate huffcr pH 8 (1:3. v/v) ins cml~loyed. :\luminuni :unmonium sulfntr, col,lwl’ and nickel c~hloritfc wrrc sulfate, cobalt chloridr, added separately to the above rewtion mistuw at different concvntrntion~ bctwwn 1 X lo-” :mtl 5 x lo-” ;vz. .\ftvr incubation, the reaction misturr ~:IP csaminrd for its ITT: absorption spwtrum. Sclxuxtion of single components of rcwtion mist,urr was whirvctl by l,nl,cr c.hromatographg in the following solwnt : ?l-prop:lnol-triethylnnlinc arc’tale buffer 0.05 151, pH 8 (4: 1). Identification of PI, and PM w:~ti accoml)lishcd in TJV light by thcit position in resprci, to samples of pure substnnccs and by the UV spectrum from cluntvs from rnch spot.
240
280
G,lBd was identified grams with ninhydrin. RESI’LTS
AID
by spraying
the chromato-
DISCUSSIOK
During incubat’ion under the above-described conditions, t’he reaction mixture containing PI, and GAB.4, both in the prescnco and absence of metal ions, becomes ycllo~v, presumably because of imine formation. The spectra of this mixture after incubation at different pH’s are represented in Fig. 1. =Zt pH 8 t’he spect,rum is very similar to that obtained from salicylaldheyde and c,t’llylcnc~dianlinc (3). This is consistent with tllc formation of a hydrogen-bonded iminc bctwcn PI, and GABA (4, 5). The prcscnce of increasing concentrations (1 X 1O-:1-5 X lo-:‘) of aluminum, copper, cobalt’, and nickel salts does not influence the cxtcnt of formation and structure of such an iminc; consequently, the absorption spclctra of the reaction mixtures are the same both in the prcsencc and absence of t11ese salts.
An increased iminc formation was obwvctl when an ethanol-phosphate buffer 0.1 M 1)H 8 (1:3, v/v) was employed (Fig. 11 (1).
360 320 Wave length, my
Fro. 1. Absorption spectra of PL (O---O), imine from PL 1 X IO-’ M and GAB$ 5 X 10-l III in aqueous buffered solution at pH 7.5 (O---O ), at pH 12.5 (~+---a), and in ethanol-phosphate buffer 0.1 fif (1:3, v/v) pH 7.5 (0-u).
PL
IMINE
PM
Acidification causes hydrolysis of irninc, as pro~cr1 by :L shift, of thv ahorption band frown the 360-430-l+ region toward that of PT,, i.c., 280-340-n1p region. Ill)011 :iddition of alkali, ionization of tllc phcnolic OH orcurs :anrl :L new ahsorption hnri wit11 :t n~axin~un~ at< 365 n+ appww, con~istr~nt with nonliydlogcn-l)ondctl iininc structure. ‘rho examination in 19’ light of the cllroinatograins rcvculs tlic prcwncc of sinall ninounts of PT, and PM anrl :I well-defined
yellow spot. Tlw sl)ot corresponding to GABA is drtcctn\)lc with ninhydrin spray (Fig. 2). Tlic spcctrunl of tlic intcriucdiate coinlwunrl r~lutcd froiu tlrrl paper is not tyl)ical of tlic pure iminr lwcause of $1tcndcncy to split, at, crluilihiulll, into PI, and GABA. Conscquent~ly, th spwtruin rc~vcals tllc, Iwescnrc of :I l~livturc containing PT, anrl iminc>. Prcscnt results gire vvidcncc for iininr fornlation froul PI, and GAB-L Ahsorption spc~ctrunl and hchavior in acid and alkalint solutions arc consistent with tlir Iiyrlrogcn-hondcri sti-u&ire. C’l~~onlatogra~)llic analysce give cvidcncc for tlic foriiiation of PM, thus inrlicating that :i partial tranl;anlination htwwn PT, ant1 (:ABA rlocs occur. hddition of rliffcwnt nlultivalcnt ions rlocs not cnhnce inlinc formation and trnnsanlination. This failure of cation action rlow not cwludc, l~o~~-cvci~, that ;I nictal iuigllt play ii role in cnzyniic transaiuination. On tlic 0th hind, tlic inactivity of tlic twhl cations iniglit 1w due to tlic structure of C;ABA in \vllich tlw carbon carrying tllc :~niino givul) is y-~:\rhon,
4. h~InwAx. Il. I<:.. .I. Am. ~‘If~~,U. sot. 79, 485 (1957). 5. l~r..\Kl.riY, 1~. I,., /lioC/lC~r/t. .I. 61, 315 (1!155).
OF
RIOCHEMISTRY
.4KD
BIOPHYSICS
On the Non-Enzymic
96, 650-652 (1962)
Transamination
c. S. ROSSI, F. OLIVO,
A. R,4BBSSISI
of r-Aminobutyric ANL)
Acid’
r\r. SII,IPR,4NDI
Investigation of chemical trnnsaminntion between pgridoxal or pyridoxnl phorIhate and the y-nminobutyric acid has been carried out,. It has thus been possible to give evidence for imine formation and for trnnsnmination betwew pyridoxal or pvridoxal phosphate and y-aminobutyric acid. Pyridoxal, iminc, and pyridoxamine have been separated by paper chromatography. Absorption spectra and behavior in acid and alkaline solutions are consistent with the hydrogen-bonded imine structure INTROD1-CTION
free phenolir group is so placed as to stabilizc the imiw through hydrogen bonding or, in tlic prescnw of appropriate metal ions, through the formation of chelatc rings:
Enzymic and non-enzymic reactions catalyzed by PLP’ are generally interpreted to occur through int’eraction between the formyl group of PL and the u-amino acid amino group with the formation of an imine : H 0 \
C-L
-0 /
II”
H I
\
CH I CHgOH II
HzC I
Bccording to Snell (1 j , in these imines a conjugate system of double bonds, extending from the a-carbon of the amino acid to the electronegative group, results in reduced electron density around t,he a-carbon atom, t’hus weakening the bonds to the carbon carrying the NH, group. In these imines, the ‘This work was supported by grants from Rockefeller Foundation and from Consiglio Sazionale dellr RicerchP. ’ A1bhrcviations used : 1’12, pyridoxal l~hor])hat~; PI,, pyridoxal ; PM, p~ridoxamin~~; C.4H.1. T-aminobutj+c avid : UV. ultraviolet 650
“Alanine-pyridoxal-Al+ + +” and “pyrurat’e-pyridoxamine-Al+ + +” imines have been recently isolated and characterized by Fasella et 01. (2). Participation of these int,ermediates in the transaminat,ion reactions was proved by the same authors. Since up to now chiefly a-amino acids haoc been considered in such “model syst~ellls,” it’ seemed interesting to investigate the possibility, extent, and mechanism of non-cnzymic transamination between PI, and a non-a-amino acid, such as GABA. In the cast of GABA, t’he amino groul) is so placed as to suggest a mechanism differcnt from thr formation of a chclate indicated as II, in which there is a bond bctwwn tlrr carboxy group and metal ion.
Moreover, the choice of this amino acid has been suggested by the particular role of GABA in cerebral metabolism in connection with PLP enzymes. MATERIALS
.\KD
METHODS
Itraction bctwwn PI, or PLI’ (2 X 10-l .l/) and GAB*1 (5 X 10-l .II) wcrc c:uri(yl out at 37°C. for dif%“crcnl t imrs. Appropriate I)uffcrs t~mployctl for the various pH’s wwc as follow: 0.05 :I! xclate at pH 4; pH 7-8, 0.05 JI phoq~hate: pH 12, 0.05 ill sodium l~ydrosidr. Altrmntivcly, nn rthanol-0.1 dl phosphate huffcr pH 8 (1:3. v/v) ins cml~loyed. :\luminuni :unmonium sulfntr, col,lwl’ and nickel c~hloritfc wrrc sulfate, cobalt chloridr, added separately to the above rewtion mistuw at different concvntrntion~ bctwwn 1 X lo-” :mtl 5 x lo-” ;vz. .\ftvr incubation, the reaction misturr ~:IP csaminrd for its ITT: absorption spwtrum. Sclxuxtion of single components of rcwtion mist,urr was whirvctl by l,nl,cr c.hromatographg in the following solwnt : ?l-prop:lnol-triethylnnlinc arc’tale buffer 0.05 151, pH 8 (4: 1). Identification of PI, and PM w:~ti accoml)lishcd in TJV light by thcit position in resprci, to samples of pure substnnccs and by the UV spectrum from cluntvs from rnch spot.
240
280
G,lBd was identified grams with ninhydrin. RESI’LTS
AID
by spraying
the chromato-
DISCUSSIOK
During incubat’ion under the above-described conditions, t’he reaction mixture containing PI, and GAB.4, both in the prescnco and absence of metal ions, becomes ycllo~v, presumably because of imine formation. The spectra of this mixture after incubation at different pH’s are represented in Fig. 1. =Zt pH 8 t’he spect,rum is very similar to that obtained from salicylaldheyde and c,t’llylcnc~dianlinc (3). This is consistent with tllc formation of a hydrogen-bonded iminc bctwcn PI, and GABA (4, 5). The prcscnce of increasing concentrations (1 X 1O-:1-5 X lo-:‘) of aluminum, copper, cobalt’, and nickel salts does not influence the cxtcnt of formation and structure of such an iminc; consequently, the absorption spclctra of the reaction mixtures are the same both in the prcsencc and absence of t11ese salts.
An increased iminc formation was obwvctl when an ethanol-phosphate buffer 0.1 M 1)H 8 (1:3, v/v) was employed (Fig. 11 (1).
360 320 Wave length, my
Fro. 1. Absorption spectra of PL (O---O), imine from PL 1 X IO-’ M and GAB$ 5 X 10-l III in aqueous buffered solution at pH 7.5 (O---O ), at pH 12.5 (~+---a), and in ethanol-phosphate buffer 0.1 fif (1:3, v/v) pH 7.5 (0-u).
PL
IMINE
PM
Acidification causes hydrolysis of irninc, as pro~cr1 by :L shift, of thv ahorption band frown the 360-430-l+ region toward that of PT,, i.c., 280-340-n1p region. Ill)011 :iddition of alkali, ionization of tllc phcnolic OH orcurs :anrl :L new ahsorption hnri wit11 :t n~axin~un~ at< 365 n+ appww, con~istr~nt with nonliydlogcn-l)ondctl iininc structure. ‘rho examination in 19’ light of the cllroinatograins rcvculs tlic prcwncc of sinall ninounts of PT, and PM anrl :I well-defined
yellow spot. Tlw sl)ot corresponding to GABA is drtcctn\)lc with ninhydrin spray (Fig. 2). Tlic spcctrunl of tlic intcriucdiate coinlwunrl r~lutcd froiu tlrrl paper is not tyl)ical of tlic pure iminr lwcause of $1tcndcncy to split, at, crluilihiulll, into PI, and GABA. Conscquent~ly, th spwtruin rc~vcals tllc, Iwescnrc of :I l~livturc containing PT, anrl iminc>. Prcscnt results gire vvidcncc for iininr fornlation froul PI, and GAB-L Ahsorption spc~ctrunl and hchavior in acid and alkalint solutions arc consistent with tlir Iiyrlrogcn-hondcri sti-u&ire. C’l~~onlatogra~)llic analysce give cvidcncc for tlic foriiiation of PM, thus inrlicating that :i partial tranl;anlination htwwn PT, ant1 (:ABA rlocs occur. hddition of rliffcwnt nlultivalcnt ions rlocs not cnhnce inlinc formation and trnnsanlination. This failure of cation action rlow not cwludc, l~o~~-cvci~, that ;I nictal iuigllt play ii role in cnzyniic transaiuination. On tlic 0th hind, tlic inactivity of tlic twhl cations iniglit 1w due to tlic structure of C;ABA in \vllich tlw carbon carrying tllc :~niino givul) is y-~:\rhon,
4. h~InwAx. Il. I<:.. .I. Am. ~‘If~~,U. sot. 79, 485 (1957). 5. l~r..\Kl.riY, 1~. I,., /lioC/lC~r/t. .I. 61, 315 (1!155).