We propose to determine a high resolution structure for KDPG aldolase with crystals grown from PEG solutions at pH 7-8 where the enzyme displays optimal catalytic activity. The active site will be located using isomorphous crystals grown with KDPGal-reduced, inactivated enzyme. Our previous work with pH 3.5 crystals did not establish the active site unequivocally. The salt-free PEG crystals will be derivatized with BrPy to locate the Glu residue which is the second nucleophile of catalysis. Our previous work placed this residue, Glu 56, about 25 A from the Schiff base forming Lys 144. This anomoly is clearly untenable. We will also increase the ionic strength of PEG crystals to fix the conformational change thought to accompany it and we will alkylate and identify the Cys which becomes activated thereof with BrPy. The Glu 56-Lys 144 anomoly will be removed independently by crosslinking these residues with BrPy and sequencing a crosslinked proteolytic peptide fragment of the enzyme. Electrophoresis of the crosslinked, reduced and """"""""dissociated"""""""" enzyme will establish whether the Glu-Lys interaction is inter-subunit. This will be confirmed by determining the activity of subunits alone. Since the pH 3.5 X-ray work suggests that the pKb of Lys 144 might be abnormally low, the latter will be determined. Lastly, comparisons of the structures of KDPG aldolase, TIM and PK, will all have the same 8-fold Alph/Beta barrel fold, will continue using the side chains of residues, particularly in the active site region.
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| Mulichak, A M; Skrzypczak-Jankun, E; Rydel, T J et al. (1988) Crystallization and preliminary diffraction data of Escherichia coli ADP glucose pyrophosphorylase. J Biol Chem 263:17237-8 |
| Skrzypczak-Jankun, E; Tulinsky, A; Fillers, J P et al. (1986) Preliminary crystallographic data and quaternary structural implications of the central subunit of the multi-subunit complex transcarboxylase. J Mol Biol 188:495-8 |
