Mapping the effector region in Thermus thermophilus elongation factor Tu.
Native elongation factor Tu from Thermus thermophilus is initially attacked by various endoproteases in a region spanning amino acid residues 40-70. By comparing the hydrolysis rates of nucleotide-free and GDP-bound EF-Tu, only a small difference was observed for the tryptic cleavage at Arg-59. Protease V-8 attacks Glu-55 only in a GDP/GTP form, whereas this enzyme exclusively hydrolyze Asn-64 in nucleotide-free EF-Tu, even when the protein had been previously cleaved at Arg-59. Binding of GDP leads to a 42-fold decreased rate of hydrolysis by the Lys-C protease at Lys-52. It also reduces the accessibility of Lys-275 to trypsin, reflecting a "long-range" effect from nucleotide binding domain I to domain II. Only slight differences were observed in the rate of hydrolysis at all positions in the GDP- versus the GTP-bound form. The intrinsic GTPase activity was slightly reduced in trypsin-treated EF-Tu, significantly impaired in EF-Tu cleaved at Lys-52, and completely abolished in EF-Tu cleaved at Asn-64. No ribosome-induced GTPase activity was observed for protease-cleaved EF-Tu's. Treatment of these proteins with periodate-oxidized GDP or GTP followed by cyanoborohydride led to covalent modification of the new N-terminus located exclusively within region 52-60. The highest reactivity was shown by the N-terminus of Glu-56. Additionally, lysine residues in the native protein sensitive to affinity labeling [Peter, M.E., Wittmann-Liebold, B., & Sprinzl, M. (1988) Biochemistry 27, 9132-9139] lost their reactivity upon cleavage of EF-Tu in region 52-60, suggesting an altered structure of the cleaved protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Laboratorium für Biochemie, Universität Bayreuth, FRG.
M E
ME
Peter
N K
NK
Schirmer
C O
CO
Reiser
M
M
Sprinzl
D000345:Affinity Labels; D000595:Amino Acid Sequence; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010447:Peptide Hydrolases; D010449:Peptide Mapping; D011487:Protein Conformation; D012270:Ribosomes / Q000378:metabolism; D013824:Thermus / Q000378:metabolism
D000345:Affinity Labels; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010447:Peptide Hydrolases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu