A conformational transition involved in antagonistic substrate binding to the allosteric phosphofructokinase from Escherichia coli.

The binding of fructose 6-phosphate, ATP or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate), ADP, and phosphoenolpyruvate to Escherichia coli phosphofructokinase has been studied by changes in the protein fluorescence and/or equilibrium dialysis. The results lead to the following conclusions: (1) tetrameric phosphofructokinase can bind four ATP but only two fructose-6-phosphate, and this binding occurs without cooperativity; (2) only two conformational states, T and R, with respectively a high and a low fluorescence, seem accessible to phosphofructokinase, which exists as a mixture of one-third R and two-third T states in the absence of ligand; (3) the substrate fructose 6-phosphate and the allosteric activator ADP bind preferentially to the low-fluorescence R state, while the other substrate, ATP [or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate)], and the allosteric inhibitor phosphoenolpyruvate bind to the high-fluorescence T state; (4) the binding of a given ligand is cooperative, with a Hill coefficient of 2, only when this binding is accompanied by a complete shift from one state to the other; for instance, the binding of the ATP analogue adenylyl 5'-(beta,gamma-methylenediphosphonate) to the T state is cooperative only in the presence of fructose 6-phosphate which favors the R state. This behavior is qualitatively consistent with a concerted transition, but quite different from that described earlier for phosphofructokinase from steady-state activity measurements (Blangy et al., 1968). This discrepancy suggests that the allosteric properties of phosphofructokinase are due in part to ligand binding and in part to the kinetics of the enzymatic reaction.     

Optical measurement of a solvent-induced isomerization in a proline-containing hexapeptide.

The hexapeptide Gly-Gly-Pro-Tyr-Gly-Gly has been synthesized and its tyrosine residue converted to nitrotyrosine by reaction with tetranitromethane. When diluted from dimethylsulfoxide into aqueous solution, the nitrated hexapeptide undergoes a slow conformational change characterized by a change in the ionization state of the nitrotyrosine group. This slow reaction is not observed with peptides containing nitrotyrosine and no proline. Also, the rate and activation enthalpy of this slow conformational change suggest that it could be due to proline cis-trans isomerization. The possibility of measuring the rate of cis-trans isomerization of proline residues in a polypeptide chain is discussed.     

A physical difference between the fast- and slow-refolding forms of nitrotyrosyl ribonuclease A: the pK values of the nitrotyrosyl groups.

In the preceding paper we present kinetic evidence for a slow equilibrium between two conformational forms of heat-unfolded ribonuclease A whose rates of refolding differ 100-fold. In a search for physical differences between these two forms, we undertook a study of the pK changes during refolding of a specific set of freely ionizing surface groups. By use of a standard procedure the three freely ionizing tyrosine groups (pK ∼- 10) have been nitrated by tetranitromethane, yielding three nitrotyrosine groups (pK ∼- 6.8). Nitrotyrosyl ribonuclease A closely resembles the unmodified enzyme as regards: (1) enzymatic activity; (2) thermal unfolding transition at neutral pH; and (3) kinetics of refolding. In particular, stopped-flow measurements of 2′CMP binding during refolding show that the fast-refolding reaction is unchanged by nitration and yields fully folded enzyme able to bind 2′CMP.The pK change of the nitrotyrosyl groups upon refolding is quite different in the fast- and slow-refolding reactions. In the slow reaction it is small (− 0.046 ± 0.006 pH unit) but easily measureable, whereas in the fast-reaction it is too small to be detected (− ΔpK less than 0.02 pH unit). This difference in pK change upon refolding can be attributed to different pK values of the nitrotyrosyl groups in the slow-refolding and fast-refolding forms of the heat-unfolded protein. Presumably the same structural differences between these two heat-unfolded forms are responsible both for the pK difference and for the 100-fold difference in rates of refolding.These results support the simple three-species mechanism for refolding discussed in the preceding paper. (a) They demonstrate a physical difference between the fast- and slow-refolding species. (b) They do not show any additional kinetic complexity when refolding is measured by a property that distinguishes between the fast- and slow-refolding species.