Does the Active Site Arginine Change the Nature of the Transition State for Alkaline Phosphatase-Catalyzed Phosphoryl Transfer?

Abstract We perform molecular dynamics simulations, based on recent crystallographic data, on the 8–17 DNAzyme at four states along the reaction pathway to determine the dynamical ensemble for the active state and transition state mimic in solution. A striking finding is the diverse roles played by Na+ and Pb2+ ions in the electrostatically strained active site that impact all four fundamental catalytic strategies, and share commonality with some features recently inferred for naturally occurring hammerhead and pistol ribozymes. The active site Pb2+ ion helps to stabilize in-line nucleophilic attack, provides direct electrostatic transition state stabilization, and facilitates leaving group departure. A conserved guanine residue is positioned to act as the general base, and is assisted by a bridging Na+ ion that tunes the pKa and facilitates in-line fitness. The present work provides insight into how DNA molecules are able to solve the RNA-cleavage problem, and establishes functional relationships between the mechanism of these engineered DNA enzymes with their naturally evolved RNA counterparts. This adds valuable information to our growing body of knowledge on general mechanisms of phosphoryl transfer reactions catalyzed by RNA, proteins and DNA.

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Faculty Opinions recommendation of Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1126855.583957 ◽

2008 ◽

Author(s):

Arieh Warshel

Keyword(s):

Transition State ◽

Magnesium Fluoride ◽

Phosphoryl Transfer ◽

Transition State Analogs ◽

Anionic Charge

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Structure and activity of phosphoglycerate mutase

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1981.0066 ◽

1981 ◽

Vol 293 (1063) ◽

pp. 121-130 ◽

Cited By ~ 70

Keyword(s):

Active Site ◽

Transfer Reaction ◽

Chemical Kinetic ◽

Phosphoglycerate Mutase ◽

Phosphoryl Transfer ◽

X Ray ◽

Histidine Residues ◽

Ping Pong ◽

Available Information ◽

Structure And Activity

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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Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin

Biochemical Journal ◽

10.1042/bj2560357 ◽

1988 ◽

Vol 256 (2) ◽

pp. 357-361 ◽

Cited By ~ 245

Author(s):

P Hadváry ◽

H Lengsfeld ◽

H Wolfer

Keyword(s):

Transition State ◽

Acid Derivative ◽

Pancreatic Lipase ◽

Active Site ◽

Boronic Acid ◽

Selective Inhibitor ◽

Covalent Intermediate ◽

State Form ◽

Covalent Inhibitor

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.

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