On the role of Glu376 in catalysis of acyl-CoA

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CU. 3 .Q ... 60. 80. 0. 11I .Q ct. Wavelength (nm). Figura l:Spectral changes observed upon incubation of LCADH with 2.4 equivalents of 2-octynoyl-CoA in 100 ...

First publ. in: Flavins and flavoproteins 1990 : proceedings of the tenth International Symposium, Como, Italy, July 15 - 20, 1990, pp. 325-328 ON THE ROLE OF GLU 376 IN CATALYSIS CATALYSIS OF OF ACYL-CoA ACYL-CoA DEHYDROGENASES Kathrin Ankele, Klaus Melde, Stefan Engst, Engst, Peter Peter Brass, Brass, Sandro Ghisla University of Konstanz PO.Box 5560 7750 Konstanz Konstanz FRG FRG Arnold W. Strauss Washington University School of Medicine, Medicine, St. St. Louis, Louis, Missouri Missouri 63110, USA Introduction: Acyl-CoA dehydrogenases are flavoproteins flavoproteins involved involved in in the the degradation of fatty acids and of branched branched chain chain amino amino acids. acids. Their reaction mechanism is assumed to involve involve aa concerted concerted n,ß-elimination, starting with abstraction abstraction of of the the a-proton a-proton [1]. [1]. Incubation of medium and short chain acyl-CoA acyl-CoA dehydrogenases dehydrogenases (MCADH and SCADH) with 2-octynoyl-CoA leads leads to to covalent covalent modification of the enzyme active site [2,3]. [2,3]. The The amino amino acid acid 376 involved is Glu , which has been proposed proposed to to be be the the base base 376 is abstracting the hydrogen as an a-proton [4]. [4]. Since Since Glu Glu376 is not not congerved in all acyl-CoA dehydrogenases dehydrogenases (Isovaleryl-CoA (Isovaleryl-CoA and and LCADH have Gly at position 376) [5], the the above above mentioned mentioned role role 376 of Glu can be questioned. In order to investigate investigate the the role role of Glu 376 , we studied the reactivity with with 2-octynoyl-CoA 2-octynoyl-CoA of of two two enzymes lacking Glu 376 . These are long chain chain acyl-CoA acyl-CoA 376-Gln dehydrogenase (LCADH) which has a Gly376 [5], [5], and and the the Glu Glu376 -Gln mutant of human MCADH [6]., Results and Discussion: Reaction of 2-octynoyl-CoA with LCADH: The The incubation incubation of of LCADH LCADH with 2-octynoyl-CoA leads to changes of the the oxidized oxidized flavin flavin spectrum which are similar to those reported reported by by Freund Freund et et ale [3] using MCADH. A difference is the complete complete

Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/5414/ Flavins 1990 Flavins and and Flavoproteins Flavoproteins 1990 http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-54140 © de & © 1991 1991 by by Walter Walter URN: de Gruyter Gruyter &Co., Co., Berlin Berlin .. New New York York -- Printed Printed in in Germany Germany

326 disappearance of the charge-transfer complex absorption in our case (see Figure 1). Importantly, however,

in the present case

the enzyme activity could be partially restored (-50%) after ultrafiltration. This indicates that 2-octynoyl-CoA does not

Figure l:Spectral changes observed upon incubation of LCADH with 2.4 equivalents of 2-octynoyl-CoA in 100 mM phosphate, pH 8, 25°C. Curves 1-3: spectra recorded at 0 mine (1), 3 mine (2), and 75 mine (3). The inset shows the formation and decay of the charge transfer complex absorption at 800 nm and the activity of the enzyme at the times shown. Note that -50 % activity could be regained after ultrafiltration.

lead to covalent modification of the active site, although it

is being acted on by the enzyme. The product acts as a strong competitive inhibitor, thus anaerobic incubation of 6 equi-

valents of octanoyl-CoA with enzyme which had been inactivated with 2-octynoyl-CoA and then ultrafiltered leads to complete reduction of the flavin spectrum within a few minutes shown) .

(data not

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Figura l:Spectral changes observed upon incubation of LCADH with 2.4 equivalents of 2-octynoyl-CoA in 100 mM phosphate, pH 8, 25°C. Curves 1-3: spectra recorded at 0 min. (1), 3 min. (2), and 75 min. (3). The inset shows the formation and decay of the charge transfer complex absorption at 800 nm and the activity of the enzyme at the times shown. Note that -50 % activity could be regained after ultrafiltration.

with 2-octynoyl-CoA and then ultrafiltered leads to complete valents of

octanoyl~CoA

with enzyme which had been inactivated

competitive inhibitor, thus anaerobic incubation of 6 equi-

is being acted on by the enzyme. The product acts as a ßtrong lead to covalent modification of the active site, although it ultrafiltration. This indicates that 2-octynoyl-CoA does not the enzyme activity could be partially restored (-50%) after

case (see Figure 1). Importantly, however, in the present case disappearance of the charge-transfer complex absorption in our 326

~ I

327

Reaction of 2-octynoyl-CoA with human Glu 376 -Gln MCADH mutant: Incubation of this human MCADH mutant with 3 equivalents of 2-octynoyl-CoA leads to spectral changes which are

2. This absorption is probably due to formation of acharge

significantly different to those observed upon incubation with

absorption at around 550 nm has developped as shown in figure

MCADH or LCADH.

(nQt shown). After 20h a distinct shoulder at 310 nm and an typical for binding of CoA-derivatives to the enzyme occurs

Immediately after addition, the red-shift of the 450 nm band 0.4

0.5

Figure 2: Spectral properties of the Glu 376 -Gln mutant of human MCADH before (1) and after 17h of aerobic incubation with 3 equivalents of 2-octynoyl-CoA (2). Spectrum (3) represents the reduced enzyme (2) after 20h of anaerobic incubation with 6.7 equivalents of octanoyl-CoA. The inset shows the spectral properties of the product of human Glu 376 -Gln MCADH mutant incubation with 2-octynoyl-CoA after ultrafiltration in 100 mM phosphate, 25 mM MgC1 2 , pH 7. At pH 5 and 9 the spectrum is unchanged (not shown). G

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Immediately after addition, the red-shift of the 450 nm band typical for binding of CoA-derivatives to the enzyme occurs (nQt shown). After 20h a distinct shoulder at 310 nm and an

MCADH or LCADH.

absorption at around 550 nm has developped as shown in figure

significantly different to those observed upon incubation with

2. This absorption is probably due to formation of acharge

2-octynoyl-CoA leads to spectral changes which are

Incubation of this human MCADH mutant with 3 equivalents of Reaction of 2-octynoyl-CoA with human Glu 376 -Gln MCADH

utant: 327

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328

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transfer complex and is similar to what is observed upon incubation of human MCADR mutant with 3-oxoacyl-CoA derivatives

Kelly,

I. Rasched, S. Ghisla (same issue).

(data not shown). The absorbance at 310 nm was decreased by

_

7.Engst, S.,P. Bross, J. Stiemke, A. Schieber, A.W. Strauss, D.

ultrafiltration. The spectrum of the product, present in the

Ghisla. 1990. J.Biol.Chem. 2Q5, 7116.

filtrate,

is compatible with a CoA-thioester (Amax 260 nm) .

6.Bross, P., S. Engst, A.W. Strauss, D.P. Kelly,

I. Rasched, S.

However, since changes in the pR (pR 5,7 and 9) have only

Chem ..

~,

16321.

little effect on the spectrum, it is unlikely that it is a

Vockley, Y. Ikeda, J. Krauss, K. Tanaka, K.. 1989. J. Biol.

3-oxoacyl-CoA ester. The enzyme recovers the ability to be

5.Matsubara, Y., Y.

Indo, E. Naito, H. Ozasa, R. Glassberg, J.

reduced by octanoyl-CoA.

4.Powell, P.J., C. Thorpe. 1988. Biochemistry 2}, 8022.

ZA, 5996. Conclusions:

3.Freund, K., J.Mizzer, W. Dick, C.Thorpe. 1985. Biochemistry 2.Fendrich, G., R. H. Abeles. 1982. Biochemistry 2l,

6685

Our results, together with those described elsewhere in this

New York, p.385.

volume for the Glu 376 -Gln mutant [7], strongly suggest that this

P.C. Engel

and S.G. Mayhew eds.) Walter de

G~uyter,

Berlin

Glu is an important, but not necessary residue for catalysis of

1.Ghisla, S. 1984. In: Flavins and Flavoproteins (R.C. Bray,

a,ß-dehydrogenation in some acyl-CoA dehydrogenases. If its role is taken over by another residue in LCADR, then this

References:

residue obviously is incapable of reacting with the products arising from octynoyl-CoA t9 form a covalent adduct.

arising from octynoyl-CoA t9 form a covalent adduct.

residue obviously is incapable of reacting with the products References:

role is taken over by another residue in LCADH, then this

1 I

a,ß-dehydrogenation in some acyl-CoA dehydrogenases. If its 1.Ghisla, S. 1984. In: Flavins and Flavoproteins

(R.C. Bray,

Glu is an important, but not necessary residue for catalysis of P.C. Engel

and S.G. Mayhew eds.) Walter de GJuyter, Berlin

volume for the Glu 376 -Gln mutant [7], strongly suggest that this New York, p.385.

Our results, together with those described elsewhere in this

2.Fendrich, G., R. H. Abeles. 1982. Biochemistry 2l,

6685

3.Freund, K., J.Mizzer, W. Dick, C.Thorpe. 1985. Biochemistry

Conclusions:

~,

5996.

4.Powell, P.J., C. Thorpe. 1988. Biochemistry 22, 8022.

reduced by octanoyl-CoA.

5.Matsubara, Y., Y.

Indo, E. Naito, H. Ozasa, R. Glassberg, J.

3-oxoacyl-CoA ester. The enzyme recovers the ability to be

Vockley, Y.

Ikeda, J. Krauss, K. Tanaka, K.. 1989. J. Biol.

little effect on the spectrum, it is unlikely that it is a

Chem ..

~,

1

16321.

However, since changes in the pH (pH 5,7 and 9) have only

6.Bross, P., S. Engst, A.W. Strauss, D.P. Kelly,

I. Rasched, S.

filtrate, is compatible with a CoA-thioester (Amax 260 nm) .

Ghisla. 1990. J.Biol.Chem. 2Q2, 7116.

ultrafiltration. The spectrum of the product, present in the

7.Engst, S.,P. Bross, J. Stiemke, A. Schieber, A.W. Strauss, D. ~

(data not shown). The absorbance at 310 nm was decreased by

Kelly,

I. Rasched, S. Ghisla (same issue) .

incubation of human MCADH mutant with 3-oxoacyl-CoA derivatives transfer complex and is similar to what is observed upon 328

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