Solution X-ray scattering studies of the yeast phosphofructokinase allosteric transition. Characterization of an ATP-induced conformation distinct in quaternary structure from the R and T states of the enzyme.

The allosteric transition of yeast phosphofructokinase has been studied by solution x-ray scattering. The scattering curves corresponding to the native enzyme (T conformation) were found to be similar to the curves recorded in the presence of saturating concentrations of fructose 6-phosphate (R conformation) or AMP (R or R' conformation). However, the curves obtained in the presence of ATP are clearly different: the radius of gyration increases and the secondary minima and maxima are systematically shifted to lower angles, suggesting a swelling of the enzyme in the presence of ATP. These results give the first direct evidence for the existence of an ATP-induced T' conformation, distinct in quaternary structure from the R and T states of the enzyme oligomer, in agreement with our previous modeling of yeast phosphofructokinase regulation. X-ray scattering data are discussed in relation to the distinct molecular mechanisms of the ATP and fructose 6-phosphate allosteric effects involving, respectively, sequential and concerted conformational changes of the enzyme oligomer.

Sulfhydryl groups of yeast phosphofructokinase-specific localization on beta subunits of fructose 6-phosphate binding sites as demonstrated by a differential chemical labeling study.

Yeast phosphofructokinase contains 83 +/- 2 cysteinyl residues/enzyme oligomer. On the basis of their reactivity toward 5,5-dithiobis(2-nitrobenzoic acid), the accessible cysteinyl residues of the native enzyme may be classified into three groups. For titrations performed with N-ethylmaleimide, subdivisional classes of reactivity are evidenced. In each case, the 6 to 8 most reactive cysteines are not protected by fructose 6-phosphate from chemical labeling and do not seem involved in subsequent enzyme inactivation. Differential labeling studies as well as direct protection experiments in the presence of fructose 6-phosphate, indicate that 12 -SH groups/enzyme oligomer (i.e. three -SH groups per binding site) are protected by the allosteric substrate from the chemical modification. Specific labeling by the differential method of the cysteinyl residues protected by fructose 6-phosphate and further separation of the two types of subunits constituting yeast phosphofructokinase, show that the substrate binding sites are localized exclusively on subunits of beta type. Thus, alpha subunits are not implicated directly in the catalytic mechanism of yeast phosphofructokinase reaction.

Specific interactions of 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase with coenzymes.

The binding of oxidized and reduced coenzyme (NAD+ and NADH) to 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD+ molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 muM at 10 degrees C, pH 7.0. The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-DADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, the reduced coenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme-NADH complex being estimated as 2.0 muM at 25 degrees C. In addition a large quenching of the NADH fluorescence (about 83%) is observed. The comparison of the dissociation constants of the coenzyme-acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes. Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.