Tài liệu Báo cáo khoa học: Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide

and protein peroxides generated by singlet oxygen attack

Philip E. Morgan1

, Roger T. Dean2 and Michael J. Davies1

1

EPR and 2

Cell Biology Groups, The Heart Research Institute, Sydney, New South Wales, Australia

Reaction of certain peptides and proteins with singlet oxygen

(generated by visible light in the presence of rose bengal dye)

yields long-lived peptide and protein peroxides. Incubation

of these peroxides with glyceraldehyde-3-phosphate dehy￾drogenase, in the absence of added metal ions, results in loss

of enzymatic activity. Comparative studies with a range of

peroxides have shown that this inhibition is concentration,

peroxide, and time dependent, with H2O2 less efficient than

some peptide peroxides. Enzyme inhibition correlates with

loss of both the peroxide and enzyme thiol residues, with a

stoichiometry of two thiols lost per peroxide consumed.

Blocking the thiol residues prevents reaction with the per￾oxide. This stoichiometry, the lack of metal-ion dependence,

and the absence of electron paramagnetic resonance (EPR)-

detectable species, is consistent with a molecular (nonradi￾cal) reaction between the active-site thiol of the enzyme and

the peroxide. A number of low-molecular-mass compounds

including thiols and ascorbate, but not Trolox C, can pre￾vent inhibition by removing the initial peroxide, or species

derived from it. In contrast, glutathione reductase and lac￾tate dehydrogenase are poorly inhibited by these peroxides

in the absence of added Fe2+–EDTA. The presence of this

metal-ion complex enhanced the inhibition observed with

these enzymes consistent with the occurrence of radical￾mediated reactions. Overall, these studies demonstrate that

singlet oxygen-mediated damage to an initial target protein

can result in selective subsequent damage to other proteins,

as evidenced by loss of enzymatic activity, via the formation

and subsequent reactions of protein peroxides. These reac￾tions may be important in the development of cellular dys￾function as a result of photo-oxidation.

Keywords: protein oxidation; protein peroxides; protein

radicals; singlet oxygen; photo-oxidation.

Singlet oxygen (molecular oxygen in its 1

Dg state; 1

O2) is

generated by a number of enzymatic and chemical reactions,

by UV exposure, and by visible light in the presence of a

number of exogenous or endogenous cellular sensitisers. 1

O2

generation has been reported in myeloperoxidase- and

eosinophil peroxidase-catalysed reactions [1–3], and by

some activated cell types including neutrophils [4], eosino￾phils [3,5], and macrophages [6]. As a result of the wide￾spread exposure of humans to UV and visible light, 1

O2 has

been suggested to play a key role in the development of a

number of human pathologies including cataract, sunburn,

some skin cancers and aging [7–12].

1

O2 reacts with a range of biological molecules including

DNA [13,14], cholesterol [15,16], lipids [15,17,18], and

amino acids and proteins [12,19,20]. Proteins are major

biological targets as a result of their abundance and high

rate constants for reaction [21], with damage occurring

primarily at Trp, Met, Cys, His and Tyr side-chains

[12,19,20]. Reaction with Trp, His and Tyr residues has

been shown to yield peroxides, although the structure of

some of these materials remains to be fully established

(reviewed in [12,19,20]). Previous studies have identified the

C-3 site on the indole ring of Trp as a major site of peroxide

formation [22], and our recent studies have demonstrated

that the major peroxide generated with Tyr residues is a

ring-derived, C-1, dieneone hydroperoxide (A. Wright,

W. A. Bubb, C. L. Hawkins & M. J. Davies, unpublished

results). Further species are also formed with free Tyr [23].

Both endo- and hydro-peroxides have been reported with

His [24]. 1

O2-mediated oxidation of proteins also yields

peroxides, with Tyr, Trp and His residues likely targets [25].

All of these peroxides are unstable in solution, with

decomposition enhanced by reducing agents, UV light and

metal ions ([25]; A. Wright, W. A. Bubb, C. L. Hawkins &

M. J. Davies, unpublished results). Reaction with some

metal ions generates radical species ([25]; A. Wright, C. L.

Hawkins & M. J. Davies, unpublished results).

Previous studies with protein peroxides generated by

high-energy radiation (e.g. c-sources, X-rays), metal ion/

peroxide systems, thermal sources of peroxyl radicals,

peroxynitrite, and activated white cells [26,27], have shown

that these species play a key role in the propagation of

oxidative chain reactions within proteins [12,28]. These

species can oxidize other biomolecules, including lipids,

Correspondence to M. J. Davies, EPR Group, The Heart Research

Institute, 145 Missenden Road, Camperdown, Sydney, New South

Wales 2050, Australia. Fax: + 61 29550 3302,

E-mail: [email protected]

Abbreviations: EPR, electron paramagnetic resonance; GAPDH,

glyceraldehyde-3-phosphate dehydrogenase; GR, glutathione reduc￾tase; GSH, reduced glutathione; LDH, lactate dehydrogenase; 2MPG,

N-(2-mercaptopropionyl)glycine; N-Ac-Trp-OMe, N-acetyl trypto￾phan methyl ester; N-Ac-Trp-OMe-OOH, peroxides formed on

N-acetyl tryptophan methyl ester by reaction with 1

O2; NEM,

N-ethylmaleimide; 1

O2, molecular oxygen in its first excited singlet

(

1

Dg) state; PBN, N-t-butyl-a-phenylnitrone.

Enzymes: glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12);

glutathione reductase (EC 1.6.4.2); lactate dehydrogenase

(EC 1.1.1.27).

Note: a website is available at www.hri.org.au

(Received 13 November 2001, revised 12 February 2002, accepted 20

February 2002)

Eur. J. Biochem. 269, 1916–1925 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02845.x