The Transition State of the Phosphoryl-Transfer Reaction Catalyzed by the Lambda Ser/Thr Protein Phosphatase

The structure of yeast phosphoglycerate mutase determined by X-ray crystallographic and amino acid sequence studies has been interpreted in terms of the chemical, kinetic and mechanistic observations made on this enzyme. There are two histidine residues at the active site, with imidazole groups almost parallel to each other and approximately 0.4 nm apart, positioned close to the 2 and 3 positions of the substrate. The simplest interpretation of the available information suggests that a ping-pong type mechanism operates in which at least one of these histidine residues participates in the phosphoryl transfer reaction. The flexible C-terminal region also plays an important role in the enzymic reaction.

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Computational insights into the S3transfer reaction: A special case of double group transfer reaction featuring bicyclically delocalized aromatic transition state geometries

Journal of Computational Chemistry ◽

10.1002/jcc.24844 ◽

2017 ◽

Vol 38 (22) ◽

pp. 1966-1973 ◽

Cited By ~ 1

Author(s):

Andrés G. Algarra

Keyword(s):

Transition State ◽

Transfer Reaction ◽

Group Transfer ◽

Special Case ◽

Double Group

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Crystal structure of Ssu72, an essential eukaryotic phosphatase specific for the C-terminal domain of RNA polymerase II, in complex with a transition state analogue

Biochemical Journal ◽

10.1042/bj20101471 ◽

2011 ◽

Vol 434 (3) ◽

pp. 435-444 ◽

Cited By ~ 27

Author(s):

Yong Zhang ◽

Mengmeng Zhang ◽

Yan Zhang

Keyword(s):

Rna Polymerase ◽

Transition State ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Complex Structure ◽

Protein Tyrosine Phosphatases ◽

Cysteine Residue ◽

Phosphoryl Transfer ◽

Terminal Domain ◽

Deep Groove

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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