Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by Formula rather than by phosphoranes

Reversible phosphorylation of the CTD (C-terminal domain) of the eukaryotic RNA polymerase II largest subunit represents a critical regulatory mechanism during the transcription cycle and mRNA processing. Ssu72 is an essential phosphatase conserved in eukaryotes that dephosphorylates phosphorylated Ser5 of the CTD heptapeptide. Its function is implicated in transcription initiation, elongation and termination, as well as RNA processing. In the present paper we report the high resolution X-ray crystal structures of Drosophila melanogaster Ssu72 phosphatase in the apo form and in complex with an inhibitor mimicking the transition state of phosphoryl transfer. Ssu72 facilitates dephosphorylation of the substrate through a phosphoryl-enzyme intermediate, as visualized in the complex structure of Ssu72 with the oxo-anion compound inhibitor vanadate at a 2.35 Å (1 Å=0.1 nm) resolution. The structure resembles the transition state of the phosphoryl transfer with vanadate exhibiting a trigonal bi-pyramidal geometry covalently bonded to the nucleophilic cysteine residue. Interestingly, the incorporation of oxo-anion compounds greatly stabilizes a flexible loop containing the general acid, as detected by an increase of melting temperature of Ssu72 detected by differential scanning fluorimetry. The Ssu72 structure exhibits a core fold with a similar topology to that of LMWPTPs [low-molecular-mass PTPs (protein tyrosine phosphatases)], but with an insertion of a unique ‘cap’ domain to shelter the active site from the solvent with a deep groove in between where the CTD substrates bind. Mutagenesis studies in this groove established the functional roles of five residues (Met17, Pro46, Asp51, Tyr77 and Met85) that are essential specifically for substrate recognition.

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ChemInform Abstract: Phosphoryl Transfer to Anionic Oxygen Nucleophiles. Nature of the Transition State and Electrostatic Repulsion.

ChemInform ◽

10.1002/chin.199001262 ◽

1990 ◽

Vol 21 (1) ◽

Author(s):

D. HERSCHLAG ◽

W. P. JENCKS

Keyword(s):

Transition State ◽

Phosphoryl Transfer ◽

Electrostatic Repulsion

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Transition-state stabilization and molecular recognition: acceleration of phosphoryl-transfer reactions by an artificial receptor

Journal of the American Chemical Society ◽

10.1021/ja00182a017 ◽

1990 ◽

Vol 112 (26) ◽

pp. 9586-9590 ◽

Cited By ~ 37

Author(s):

Paolo Tecilla ◽

Suk Kyu Chang ◽

Andrew D. Hamilton

Keyword(s):

Molecular Recognition ◽

Transition State ◽

Transfer Reactions ◽

Phosphoryl Transfer ◽

Transition State Stabilization ◽

Artificial Receptor

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Alkali metal ion catalysis in nucleophilic displacement by ethoxide ion on p-nitrophenyl phenylphosphonate: Evidence for multiple metal ion catalysis

Canadian Journal of Chemistry ◽

10.1139/v02-202 ◽

2003 ◽

Vol 81 (1) ◽

pp. 53-63 ◽

Cited By ~ 37

Author(s):

Erwin Buncel ◽

Ruby Nagelkerke ◽

Gregory RJ Thatcher

Keyword(s):

Alkali Metal ◽

Transition State ◽

Metal Ions ◽

Metal Ion ◽

Alkali Metal Ions ◽

Phosphoryl Transfer ◽

Alkali Metal Ion ◽

Nucleophilic Displacement ◽

Displacement Reactions ◽

Metal Ion Catalysis

In continuation of our studies of alkali metal ion catalysis and inhibition at carbon, phosphorus, and sulfur centers, the role of alkali metal ions in nucleophilic displacement reactions of p-nitrophenyl phenylphosphonate (PNPP) has been examined. All alkali metal ions studied acted as catalysts. Alkali metal ions added as inert salts increased the rate while decreased rate resulted on M+ complexation with 18-crown-6 ether. Kinetic analysis indicated the interaction of possibly three potassium ions, four sodium ions, and five lithium ions in the transition state of the reactions of ethoxide with PNPP. Pre-association of the anionic substrate with two metals ions in the ground state gave the best fit to the experimental data of the sodium system. Thus, the study gives evidence of the role of several metal ions in nucleophilic displacement reactions of ethoxide with anionic PNPP, both in the ground state and in the transition state. Molecular modeling of the anionic transition state implies that the size of the monovalent cation and the steric requirement of the pentacoordinate transition state are the primary limitations on the number of cations that can be brought to bear to stabilize the transition state and catalyze nucleophilic substitution at phosphorus. The bearing of the present work on metal ion catalysis in enzyme systems is discussed, in particular enzymes that catalyze phosphoryl transfer, which often employ multiple metal ions. Our results, both kinetic and modeling, reveal the importance of electrostatic stabilization of the transition state for phosphoryl transfer that may be effected by multiple cations, either monovalent metal ions or amino acid residues. The more such cations can be brought into contact with the anionic transition state, the greater the catalysis observed.Key words: alkali metal ion catalysis, nucleophilic displacement at phosphorus, multiple metal ion catalysis, phosphoryl transfer.

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