Pyruvate site of pyruvate phosphate dikinase: crystal structure of the enzyme-phosphonopyruvate complex, and mutant analysis.
Crystals of pyruvate phosphate dikinase in complex with a substrate analogue inhibitor, phosphonopyruvate (K(i) = 3 microM), have been obtained in the presence of Mg(2+). The structure has been determined and refined at 2.2 A resolution, revealing that the Mg(2+)-bound phosphonopyruvate binds in the alpha/beta-barrel's central channel, at the C-termini of the beta-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the 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.
Herzberg
Osnat
O
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA. osnat@carb.nist.gov
Chen
Celia C H
CC
Liu
Sijiu
S
Tempczyk
Aleksandra
A
Howard
Andrew
A
Wei
Min
M
Ye
Dongmei
D
Dunaway-Mariano
Debra
D
D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011772:Pyruvate, Orthophosphate Dikinase / Q000737:chemistry* / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001051:Apoenzymes; D011994:Recombinant Proteins; D019289:Pyruvic Acid; D011772:Pyruvate, Orthophosphate Dikinase