Pyruvate Site of Pyruvate Phosphate Dikinase: Crystal Structure of the Enzyme−Phosphonopyruvate Complex, and Mutant Analysis†,‡

Osnat Herzberg, Celia C. H. Chen, Sijiu Liu, Aleksandra Tempczyk, Andrew Howard, Min Wei, Dongmei Ye, Debra Dunaway-Mariano
2002 Biochemistry  
Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, phosphonopyruvate (K i ) 3 µM), have been obtained in the presence of Mg 2+ . The structure has been determined and refined at 2.2 Å resolution, revealing that the Mg 2+ -bound phosphonopyruvate binds in the R/ -barrel's central channel, at the C-termini of the -strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but
more » ... e structure (but not sequence homology) to pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK-phosphonopyruvate complex and the structures of two complexes of pyruvate kinase, one with Mg 2+ -bound phospholactate and the other with Mg 2+ -oxalate and ATP, revealed that the two enzymes share some key features that facilitate common modes of substrate binding. There are also important structural differences; most notably, the machinery for acid/base catalysis is different. PPDK 1 catalyzes the interconversion of ATP, P i , and pyruvate, with AMP, PP i , and PEP, in three Mg 2+ -dependent partial reactions that involve phospho-and pyrophosphoenzyme intermediates (1, 2): †
doi:10.1021/bi011799+ pmid:11790099 fatcat:dvzggrjqivhuhopoirskgtnnti