Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome.
Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis. The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood. It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis. However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified. Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10(6)-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly. These results show that His84 plays a key role in the chemical step of GTP hydrolysis. Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPgammaS, showed no dependence on pH, indicating that His84 does not act as a general base. We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the gamma-phosphate group of GTP.
Daviter
Tina
T
Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer Strasse 10, 58448, Witten, Germany
Wieden
Hans-Joachim
HJ
Rodnina
Marina V
MV
D001665:Binding Sites; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D010455:Peptides; D012360:RNA, Transfer, Phe; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu