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

FEBS Letters

Krab, Ivo M.²  Parmeggiani, Andrea²  Scarano, Giuliana²  Bocchini, Vincenzo¹ 

[1]Dipartmento di Biochimica e Biotecnologie mediche, Universitá di Napoli Federico II, Via S. Pansini 5, I-80131 Napoli, Italy

[2]S.D.I. no. 61840 du Centre National de la Recherche Scientifique, Laboratoire de Biochimie, Ecole Polytechnique, F-91128 Palaiseau Cedex, France

关键词: Protein biosynthesis;    GTPase;    GTP-binding protein;    Elongation factor Tu;    Site-directed mutagenesis;    EF;    elongation factor;    ME;    2-mercaptoethanol;    DTT;    dithiothreitol;    GST;    glutathione-S-transferase;    GMPPNP;    guanosine 5′-[β;    γ-imido]triphosphate;    PEP;    phosphoenolpyruvate;    PK;    pyruvate kinase;    IPTG;    isopropyl-ß-d-thiogalactopyranoside;    Pi;    inorganic phosphate;    TCA;    trichloroacetic acid;   

DOI  :  10.1016/0014-5793(95)00469-P

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Substitution of His-84 (→ Gln 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-Tu 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-Tu 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.

【 授权许可】

Unknown