The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2.
A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.
Panniers
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University of Rochester Cancer Center, New York 14642.
Rowlands
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Henshaw
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D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D010448:Peptide Initiation Factors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism* / Q000494:pharmacology*; D012358:RNA, Transfer, Met / Q000378:metabolism; D013696:Temperature
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D006150:Guanine Nucleotides; D010448:Peptide Initiation Factors; D011506:Proteins; D012358:RNA, Transfer, Met; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium