Tài liệu Báo cáo khoa học: Site-directed mutagenesis of a loop at the active site of E1 (a2b2) of

Site-directed mutagenesis of a loop at the active site of E1 (a2b2)

of the pyruvate dehydrogenase complex

A possible common sequence motif

Markus Fries*, Hitesh J. Chauhan†

, Gonzalo J. Domingo‡

, Hyo-Il Jung§ and Richard N. Perham

Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK

Limited proteolysis of the pyruvate decarboxylase (E1, a2b2)

component of the pyruvate dehydrogenase (PDH) multi￾enzyme complex ofBacillus stearothermophilus has indicated

the importance for catalysis of a site (Tyr281-Arg282) in the

E1a subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. &

Perham, R.N. (2000) Eur. J. Biochem. 267, 7158–7169). This

site appears to be conserved in the a-subunit of hetero￾tetrameric E1s and multiple sequence alignments suggest

that there are additional conserved amino-acid residues in

this region, part of a common pattern with the consensus

sequence -YR-H-D-YR-DE-. This region lies about 50

amino acids on the C-terminal side of a 30-residue motif

previously recognized as involved in binding thiamin

diphosphate (ThDP) in all ThDP-dependent enzymes. The

role of individual residues in this set of conserved amino

acids in the E1a chain was investigated by means of site￾directed mutagenesis. We propose that particular residues

are involved in: (a) binding the 2-oxo acid substrate,

(b) decarboxylation of the 2-oxo acid and reductive acety￾lation of the tethered lipoyl domain in the PDH complex,

(c) an
open–close mechanism of the active site, and

(d) phosphorylation by the E1-specific kinase (in eukaryotic

PDH and branched chain 2-oxo acid dehydrogenase com￾plexes).

Keywords: pyruvate dehydrogenase; multienzyme complex;

thiamin diphosphate; limited proteolysis; enzyme mecha￾nism.

The family of 2-oxo acid dehydrogenase (2-OADH) multi￾enzyme complexes contains three members: the pyruvate

dehydrogenase (PDH), the 2-oxoglutarate dehydrogenase

(OGDH) and the branched-chain 2-oxo acid dehydrogenase

(BCDH) complexes. These are responsible for the oxidative

decarboxylation of pyruvate, 2-oxoglutarate and branched￾chain 2-oxo acids, respectively, in each case generating the

corresponding acyl-CoA and NADH. The complexes all

occupy important positions in the metabolism of the cell

and generally comprise three component enzymes: a 2-oxo

acid decarboxylase (E1, EC 1.2.4), a dihydrolipoyl acyl￾transferase (E2, EC 2.3.1), and dihydrolipoyl dehydroge￾nase (E3, EC 1.8.1.4). E1 catalyses the first and irreversible

step of the overall reaction, the thiamin-diphosphate

(ThDP)-dependent oxidative decarboxylation of the 2-oxo

acid, followed by the reductive acylation of a lipoyl

prosthetic group covalently bound to a lysine residue in

the lipoyl domain of the E2 chain. The reaction catalysed by

E1 is rate-limiting for the overall activity of the complex

[1,2], probably at the reductive acylation step [2,3]. The E2

component catalyses the transfer of the acyl group from the

lipoyl-lysine group to CoA, and the cycle is completed by

reoxidation of the resulting dihydrolipoyl group catalysed

by E3, a flavoprotein, generating NADH and H+ from

NAD+. For recent reviews, see de Kok et al. and Perham

[4,5].

In eukaryotic PDH and BCDH complexes, modulation

of catalytic activity is achieved by phosphorylation￾dephosphorylation of E1; an E1-specific kinase inactivates

E1 by phosphorylating certain serine residues in the E1a

component and activity is restored by the action of a specific

phosphatase [6–8]. Depending on the organism and the type

of 2-OADH complex, the E1 component exists either as a

heterotetramer (a2b2) or a homodimer (a2). In the PDH

complexes of Gram-negative bacteria and in all OGDH

complexes, E1 is a homodimer; in PDH complexes of

Correspondence to R. N. Perham, Department of Biochemistry,

University of Cambridge, Sanger Building, Old Addenbrooke’s Site,

80 Tennis Court Road, Cambridge CB2 1GA, UK.

Fax: + 44 1223 333667, Tel.: + 44 1223 333663,

E-mail: [email protected]

Abbreviations: BCDH, branched-chain 2-oxo acid dehydrogenase;

DCPIP, 2,6-dichlorophenolindophenol; E1, 2-oxo acid decarboxylase;

E1a, alpha subunit of E1; E1b, beta subunit of E1; E1p, pyruvate

decarboxylase of PDH complex; E2p, dihydrolipoyl acetyltransferase;

E3, dihydrolipoyl dehydrogenase; IPTG, isopropyl thio-b-D-galacto￾side; OGDH, 2-oxoglutarate dehydrogenase; PSBD, peripheral

subunit-binding domain; SPR, surface plasmon resonance;

ThDP, thiamin diphosphate.

Enzymes: pyruvate decarboxylase (EC 1.2.4.1); dihydrolipoyl acetyl￾transferase (EC 2.3.1.12); dihydrolipoyl dehydrogenase (EC 1.8.1.4).

*Present address: Cambridge Institute for Medical Research,

Wellcome Trust/MRC Building, Box139 Addenbrooke’s Hospital,

Hills Road, Cambridge CB2 2XY, UK.

Present address: Adprotech Ltd, Chesterford Research Park, Little

Chesterford, Saffron Walden, Essex CB10 1XL, UK.

Present address: Seattle Biomedical Research Institute, 4 Nickerson

Street, Seattle, WA 98109, USA.

§Present address: Wolfson Institute for Biomedical Research, The

Cruciform Building, Gower Street, London WC1E 6BT, UK.

(Received 19 September 2002, revised 5 December 2002,

accepted 20 December 2002)

Eur. J. Biochem. 270, 861–870 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03444.x