Toward a Quantum-Mechanical Description of Metal-Assisted Phosphoryl Transfer in Pyrophosphatase

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the yeast PPase product complex at 1....

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2001-03, Vol.98 (6), p.3121-3126
Main Authors: Heikinheimo, P., Tuominen, V., A.-K. Ahonen, Teplyakov, A., Cooperman, B. S., Baykov, A. A., Lahti, R., Goldman, A.
Format: Article
Language:English
Subjects:
Active sites
Atoms
Biochemistry
Biological Sciences
Catalysis
Coordination polymers
Crystallography, X-Ray
Diphosphates - chemistry
Enzymes
Fluorides - chemistry
Hydrogen bonds
Kinetics
Metals
Molecules
Nucleophiles
Phosphates
Phosphorus - chemistry
Protein Structure, Tertiary
Pyrophosphatases - chemistry
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Summary:The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (Onu) directly attacks PPi with a pKa vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and Onu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.061612498