The effect of hinge mutations on effector binding and domain rotation in Escherichia coli D-3-phosphoglycerate dehydrogenase

Sanghamitra Dey, Zhiqin Hu, Lan Xu Xiao, James C. Sacchettini, Gregory A. Grant

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15 Scopus citations


D-3-Phosphoglycerate dehydrogenase (EC from Escherichia coli contains two Gly-Gly sequences that have been shown previously to have the characteristics of hinge regions. One of these, Gly336-Gly 337, is found in the loop between the substrate binding domain and the regulatory domain. Changing these glycine residues to valine affected the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity was caused primarily by a decrease in the affinity of the enzyme for L-serine. These mutations also affected the domain rotation of the subunits in response to L-serine binding. A major conclusion of this study was that it defines a minimal limit on the necessary conformational changes leading to inhibition of enzyme activity. That is, some of the conformational differences seen in the native enzyme upon L-serine binding are not critical for inhibition, whereas others are maintained and may play important roles in inhibition and cooperativity. The structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6° in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer. Moreover the structures show that both pairs of Asn190 to Asn190 hydrogen bonds across the subunit interfaces are necessary for activity. These observations are consistent with the half-the-sites activity, flip-flop mechanism proposed for this and other similar enzymes and suggest that the Asn190 hydrogen bonds may function in the conformational transition between alternate half-the-site active forms of the enzyme.

Original languageEnglish
Pages (from-to)18418-18426
Number of pages9
JournalJournal of Biological Chemistry
Issue number25
StatePublished - Jun 22 2007


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