tailieunhanh - Báo cáo khoa học: Correlation between conformational stability of the ternary enzyme–substrate complex and domain closure of 3-phosphoglycerate kinase

3-Phosphoglycerate kinase (PGK) is a typical two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain–domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. | ềFEBS Journal Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase ZknHros Varna1 Roĩá a Flarhnor1 C o rírárĩor1 Clcx ath2 Androa N R7Ìlán i1 HnJica Volga LJcata Iiauiii lei LVa eidUzci ozauuius usvaui i luica IM. uziiagyi and Maria Vas1 1 Institute of Enzymology BiologicalResearch Center Hungarian Academy of Sciences Budapest Hungary 2 Department of Biophysics and Radiation Biology Semmelweis University Budapest Hungary Keywords domain closure phosphoglycerate kinase molecular graphics substrate effect thermal unfolding Correspondence M. Vas Institute of Enzymology BRC Hungarian Academy of Sciences H-1518 Budapest PO Box 7 Hungary Fax 36 1466 5465 Tel 36 1279 3152 E-mail vas@ Received 18 December 2004 revised 15 February 2005 accepted 17 February 2005 doi 3-Phosphoglycerate kinase PGK is a typical two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain-domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N- or in the C-terminal domain were also studied. The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous highly cooperative unfolding of the two domains. This cooperativity is preserved in the presence of substrates 3-phosphoglycerate bound to the N domain or the nucleotide MgADP MgATP bound to the C domain increased the structural stability of the whole molecule. A structural explanation of domain-domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures. Well-defined backbone and .