Unisite ATP hydrolysis by soluble Rhodospirillum rubrum F1–ATPase is accelerated by Ca2+

AbstractAt saturating concentrations of ATP, soluble F1 from the Rhodospirillum rubrum (RF1) exhibits a higher rate of hydrolysis with Ca2+ than with Mg2+. The mechanisms involved in the expression of a higher catalytic activity with Ca2+ were explored by measuring the ATPase activity of RF1 at substiochiometric concentrations of ATP (unisite conditions). At a ratio of 0.25 [γ-32P]ATP per RF1, the enzyme exhibited a 50 times higher hydrolytic rate with Ca2+ than with Mg2+. The rate of [γ-32P]ATP binding to RF1 was in the same range with the two divalent metal ions. Centrifugation–filtration of RF1 exposed to substoichiometric [γ-32P]ATP concentrations and Mg2+ through Sephadex columns yielded an enzyme that contained [γ-32P]ATP and [32P]phosphate in a stoichiometry that was close to one. In the presence of Ca2+, the eluted enzyme did not contain [γ-32P]ATP nor [32P]phosphate. This indicated that the rate of product release was faster with Ca2+ than with Mg2+. It was also observed that the ratio of multisite to unisite hydrolysis rates was of similar magnitude with both divalent cations. This suggests that they do not affect differently the cooperative mechanisms that may exist between catalytic sites. In consequence, the higher ATPase activity of RF1 in presence of Ca2+ strongly suggests that the retention time of products is decreased in the presence of this cation

A Ribosomal Misincorporation of Lys for Arg in Human Triosephosphate Isomerase Expressed in Escherichia coli Gives Rise to Two Protein Populations

We previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame