Relevance of histidine‐84 in the elongation factor Tu GTPase activity and in poly(Phe) synthesis: Its substitution by glutamine and alanine

Abstract

Substitution of His-84 (-~ Gin and -~ Ala), a residue of the switch II region of E. coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP. The activity in poly(Phe) synthesis and GTP hydrolysis of EF-Tu H84Q were both reduced to about 35%, as compared to EF-Tu wt, whereas EF-Tn H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity. Phe-tRNA Phe exerted a similar inhibitory effect on the GTPase activity of EF-Tn wt and EF-Tu H84Q while abolishing that of EF-Tu H84A. Ribosomes enhanced the GTPase activity of EF-Tu H84Q, but not that of EF-Tu H84A, on which they even seemed to exert an inhibitory effect. The one-round GTP hydrolysis associated with the EF-TuH84Q-dependent binding of Phe-tRNA phe to poly(U)-programmed ribosomes was less efficient than with EF-Tu wt. Kirromycin stimulated the GTPase activities of both mutants less than EF-Tu wt. The results of this work do not support a catalytic role of His-84 in the intrinsic GTPase of EF-Tu, but they emphasize the importance of its side-chain for polypeptide synthesis and GTP hydrolysis. a residual GTPase activity, making it unlikely that H84 is solely responsible for the catalysis [5].In order to shed further light on its function, His-84 has been substituted by glutamine or alanine in the intact EF-Tu. The former substitution introduces the same residue as found in the homologous position in p21, which shares several properties with histidine including the ability of forming a hydrogen bond; the latter one investigates the effects due to the elimination of interactions but the hydrophobic ones. We did not introduce a glycine, as in the case of the G domain H84G, to avoid possible side-effects associated with the increased backbone flexibility of this residue.The results show that the substitution H84Q is compatible with polypeptide synthesis and GTPase activity, whereas EFTu H84A can only sustain a low intrinsic GTPase activity. This emphasizes the importance of the nature of the side-chain in position 84 for a physiological function of EF-Tu in protein biosynthesis and the associated hydrolysis of GTP.

Limited Proteolysis and Amino Acid Replacements in the Effector Region ofThermus thermophilusElongation Factor Tu

“…GuoPP[NH]P Xray structure, it was suggested that a catalytic triad similar to that found in serine proteases, which involves Asp87, His85 and the water molecule H2041 1, leads to formation of a hydroxyl anion which then attacks the y-phosphate of GTP (Berchthold et al, 1993). In agreement with such a mechanism, the replacement of His85 by Leu or Ala renders EF-Tu almost completely inactive with regard to intrinsic and ribosome-stimulated CTPase activity (Zeidler et a]., 1995;Scarano et al, 1995). Conformational changes in the EF-Tu .…”

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

“…However, in the present case, we have a hint from direct experiments. That is, experimental studies of the H84Q mutation in Thermus thermophilus (23,25) revealed that this mutation leads only to a small reduction in the GTP hydrolysis rate (i.e., in the presence of a ribosome and the aa-tRNA) relative to the native enzyme. Unfortunately, in the T. thermophilus system studied in refs.…”

“…However, despite major biochemical and structural breakthroughs, a detailed explanation of how the codon recognition in the 30S subunit leads to GTP hydrolysis remains elusive. That is, although it is known that the precise positioning of H84 is critical for efficient catalysis (1,9,(11)(12)(13)(23)(24)(25)(26), the energetics of this positioning and its ultimate role in stabilizing the transition state (TS) are unclear. More precisely, it is frequently assumed that the conserved H84 is moved to a catalytic configuration and then serves as a general base, but this assumption is problematic (see ref.…”

“…Breakthrough in the elucidation of the ribosome structure (2-13) and careful biochemical studies (1)(2)(3)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) allow one to begin thinking about the nature of the mechanism of the elongation process. In particular, the elucidation of structure of the EF-Tu/ribosome complex (our study refers to the structure of EF-Tu in the complex as EF-Tu′) (9) opens the way for the exploration of the activation process at the molecular level.…”

Energetics of activation of GTP hydrolysis on the ribosome

“…The translational GTPases are generally poor catalysts of hydrolysis by themselves, but become activated for rapid catalysis in complex with the ribosome. The process by which the GTPases are activated has been enigmatic, although components such as the universally conserved ribosomal sarcinricin loop (SRL) and invariant His84 (Escherichia coli EF-Tu numbering) of the translational GTPases have been identified as being important for GTPase activation [3][4][5][6][7] .…”