Role of side-chains in the operation of the main molecular hinge of 3-phosphoglycerate kinase

Szabó, Judit; Varga, Andrea; Flachner, Beáta; Konarev, Peter V.; Svergun, Dmitri I.; Závodszky, Péter; Vas, Mária

doi:10.1016/j.febslet.2008.03.016

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Volume 582, Issue 9 , Pages 1335-1340, 16 April 2008

Role of side-chains in the operation of the main molecular hinge of 3-phosphoglycerate kinase

Edited by Miguel De la Rosa

Judit Szabó
- Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary
- These authors contributed equally to the work.
Andrea Varga
- Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary
- These authors contributed equally to the work.
Beáta Flachner
- Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary
Peter V. Konarev
- EMBL Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany
- Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia
Dmitri I. Svergun
- EMBL Outstation, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany
- Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia
Péter Závodszky
- Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary
Mária Vas
- Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary
- Corresponding author. Fax: +36 1 466 5465.

Received 30 January 2008; received in revised form 14 February 2008; accepted 12 March 2008. published online 20 March 2008.

Abstract

The single mutants (F165A, E192A, F196A, S392A, T393A) at and near the main hinge (β-strand L) of human 3-phosphoglycerate kinase (hPGK) exhibit variously reduced enzyme activity, indicating the cumulative effects of these residues in regulating domain movements. The residues F165 and E192 are also essential in maintaining the conformational integrity of the whole molecule, including the hinge-region. Shortening of βL by deleting T393 has led to a dramatic activity loss and the concomitant absence of domain closure (as detected by small angle X-ray scattering), demonstrating the role of βL in functioning of hPGK. The role of each residue in the conformational transmission is described.

Abbreviations: AMP-PNP, β,γ-imido-adenosine-5′ triphosphate, 3-PG, 3-phosphoglycerate, 1,3-BPG, 1,3-bisphosphoglycerate, CD, circular dichroism, DSC, differential scanning calorimetry, Nbs₂, Ellman’s reagent, 5,5′-dithiobis-(2-nitrobenzoic acid), PGK, 3-phospho-d-glycerate kinase (EC 2.7.2.3), hPGK, human PGK, SAXS, small angle X-ray scattering