Site‐Directed Mutagenesis of <i>Thermus thermophilus</i> Elongation Factor Tu

Zeidler, Waltraud; Egle, Christian; Ribeiro, Sofia; Wagner, Annett; Katunin, Vladimir I.; Kreutzer, Roland; Rodnina, Marina V.; Wintermeyer, Wolfgang; Sprinzl, Mathias

doi:10.1111/j.1432-1033.1995.0596j.x

Cited by 5 publications

(3 citation statements)

References 48 publications

(78 reference statements)

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“…In the case of EF-Tu and EF-G, on the basis of crystallographic evidence, this histidine has been argued to be the general base in the reaction . In the case of Ras, experimental data on the catalytic activity of this enzyme variant is available for only the Ras-catalyzed reaction; however, curiously, both the Q61H substitution in Ras and the H84Q substitution in EF-Tu have been shown to be detrimental to the catalytic activity of the corresponding enzyme, and therefore, the residues at these positions are not interchangeable. In the case of Ras, if Q61 were acting as a general base, as has been suggested in the literature, ,,,, one would expect a histidine rather than a glutamine at position 61 to be more catalytically favorable, as a histidine side chain is a much better candidate for a general base than the Q61 amide group, due to the substantially higher p K a of the imidazole.…”

Section: Results and Discussionmentioning

confidence: 99%

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases

et al. 2019

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GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the Gαi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4 – and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) “undruggable” GTPases like Ras.

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Section: Results and Discussionmentioning

confidence: 99%

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases

et al. 2019

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“…Due to the neutral pK a of histidine in solution and its position close to the catalytic water molecule, as seen from the structure of the ribosome‐bound EF‐Tu in the pre‐hydrolysis state (Figure b), His84 was proposed to act as a general base . This model was initially supported by the finding that substitution of His84 with a glutamine (His84Gln), another good proton acceptor, does not completely abolish the GTPase activity of EF‐Tu . However, biochemical data showed that hydrolysis of GTP and of the slowly hydrolysable analog GTPγS on the ribosome is not pH‐dependent over the neutral range, where the side chain of His84 is expected to ionize; thus, the general base mechanism seems unlikely at least in the presence of the ribosome .…”

Section: Multiple Pathways Of Gtp Hydrolysismentioning

confidence: 99%

“…Biochemical studies of EF‐Tu guided by structures and comparisons to other GTPases identified two key residues that are required for its GTPase activity on the ribosome, His84 and Asp21 . His84 in the switch II is conserved in all trGTPases.…”

Section: Multiple Pathways Of Gtp Hydrolysismentioning

confidence: 99%

Review: Translational GTPases

Maracci

Rodnina

2016

Biopolymers

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Translational GTPases (trGTPases) play key roles in facilitating protein synthesis on the ribosome. Despite the high degree of evolutionary conservation in the sequences of their GTP‐binding domains, the rates of GTP hydrolysis and nucleotide exchange vary broadly between different trGTPases. EF‐Tu, one of the best‐characterized model G proteins, evolved an exceptionally rapid and tightly regulated GTPase activity, which ensures rapid and accurate incorporation of amino acids into the nascent chain. Other trGTPases instead use the energy of GTP hydrolysis to promote movement or to ensure the forward commitment of translation reactions. Recent data suggest the GTPase mechanism of EF‐Tu and provide an insight in the catalysis of GTP hydrolysis by its unusual activator, the ribosome. Here we summarize these advances in understanding the functional cycle and the regulation of trGTPases, stimulated by the elucidation of their structures on the ribosome and the progress in dissecting the reaction mechanism of GTPases. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 463–475, 2016.

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Site‐directed mutagenesis of Thermus thermophilus EF‐Tu: the substitution of threonine‐62 by serine or alanine

et al. 1995

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The invariant threonine-62, which occurs in the effector region of all GTP/GDP-binding regulatory proteins, was substituted via site-directed mutagenesis by alanine and serine in the elongation factor Tu from Thermus thermophilus. The altered proteins were overproduced in Escherichia coli, purified and characterized. The EF-Tu T62S variant had similar properties with respect to thermostability, aminoacyl-tRNA binding, GTPase activity and in vitro translation as the wild-type EF-Tu. In contrast, EF-Tu T62A is severely impaired in its ability to sustain polypeptide synthesis and has only very low intrinsic and ribosome-induced GTPase activity. The affinity of aminoacyl-tRNA to the EF-Tu T62A.GTP complex is almost 40 times lower as compared to the native EF-Tu.GTP. These observations are in agreement with the tertiary structure of EF-Tu.GTP, in which threonine-62 is interacting with the Mg2+ ion, gamma-phosphate of GTP and a water molecule, which is presumably involved in the GTP hydrolysis.

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Site‐Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu

Cited by 5 publications

References 48 publications

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases

Review: Translational GTPases

Site‐directed mutagenesis of Thermus thermophilus EF‐Tu: the substitution of threonine‐62 by serine or alanine

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