François Seydoux

Binding of nucleotides AMP and ATP to yeast phosphofructokinase:evidence for distinct catalytic and regulatory subunits.

Abstract The binding of ATP to yeast phosphofructokinase, as monitored by flow dialysis, is heterogeneous and is adequately described by assuming two independent classes of three binding sites each per enzyme molecule. Under similar conditions, the binding of 5′AMP is homogeneous and a binding stoichiometry of three 5′AMP molecules per enzyme molecule is evaluated. Displacement experiments show that only the ATP molecules bound to the first class of three tight binding sites are displaced by an excess of 5′AMP. Thus, these tight ATP binding sites can be identified as “regulatory sites” in agreement with kinetic data. Furthermore, molecular weight determinations and electrophoresis results are consistent with an heterologous α 3 β 3 structure of the enzyme oligomer. Therefore the present binding data suggest that yeast phosphofructokinase is constituted by three “catalytic” and three “regulatory” subunits.

Pseudoconservative transition: A two-state model for the co-operative behavior of oligomeric proteins☆

Abstract A plausible extension of the two-state concerted model is proposed. This extended two-state model involves at least one state with pairwise asymmetry and consequent heterogeneous binding properties. Such a pseudoconservative transition model accounts in a predictably restricted manner for either positive or negative co-operativity † and for a combination of both in the binding of a single ligand. State and saturation functions are derived. Variation of the Hill number with respect to the extent of binding provides a diagnostic test for the combination of both positive and negative co-operativities in ligand binding. Applications of the model to recent experimental data are discussed.

Influence of phosphate on the allosteric behavior of yeast phosphofructokinase

Abstract Initial rate data obtained with purified yeast phosphofructokinase (PFK) show an ATP dependent kinetic cooperativity with respect to fructose-6-phosphate. In the presence of 25 mM phosphate, the cooperativity index (Hill number) is related to the half saturation concentration of fructose-6-phosphate as predicted by the concerted allosteric model in the case of a “K-system”. In the absence of phosphate, however, the kinetic behavior of yeast PFK is more complex and the cooperativity index is invariant with respect to the half saturation concentration of fructose-6-phosphate which is increased by ATP. In both cases, 5′AMP behaves as a strong activator of the enzyme. These kinetic data suggest that the two distinct functions of ATP as phosphate donnor and as allosteric inhibitor, respectively, are supported by different binding sites. These regulatory properties of yeast PFK are discussed in relation to glycolytic oscillations.

Octameric structure of yeast phosphofructokinase as determined by crosslinking with disuccinimidyl β‐hydromuconate

Yeast phosphofructo~nase (EC 2.7.1.1 l), a key enzyme in the regulation of the glycolytic pathway, is built up of an equal number of two types of subunits (Y and /3 which are immunologically distinct [l] and further differentiated by their relative susceptibility to proteolytic degradation in the presence of specific ligands [2]. Binding experiments [3,4] as well as functional properties of yeast phosphofructokinase [4,5] were indicative of a small number of interacting protomers (3 or 4) equal to only half the number of subunits constituting the enzyme oligomer. However, quaternary structure of yeast phosphofructokinase is still a matter of controversy since molecular weight determinations were unable to discriminate unambiguously between a hexameric and an octameric structure of the enzyme [4,6,7]. Thus, the major evidence for an octameric structure was obtained from small-angle X-ray scattering [S] and from crosshnking experiments with dimethyl suberimidate [9]. However, these data could not rule out an eventual hexameric structure for yeast phosphofructokinase. Indeed, the interpretation of X-ray diffusion data relies heavily on the assumption of an arbitrary chosen shape of the subunits. Moreover, no polymeric forms higher than tetramer

On the specificity of tryptic catalysis

Summary The specificity of esterasic tryptic action is discussed in terms of the formation of two intermediates (Michaelis complex and acylenzyme) during the catalytic process. From nucleophilic competition data with specific and unspecific substrates and some other complementary data, it is concluded that the tryptic specificity is essentially kinetic and exerted principally in the acylation step. Thus discrimination between specific and unspecific substrates is achieved also after the Michaelis complex formation and is not only reflected by the binding free enthalpy but also by the free enthalpy change associated with the acylation of the enzyme.

Studies on the structure of yeast phosphofructokinase

In this paper, we describe an efficient procedure for the purification of yeast phosphofructokinase. This procedure eliminates any time delay and enables to obtain an enzyme with minimum proteolytic alterations. The molecular weights of the oligomeric enzyme and of its constitutive subunits were both evaluated by means of several independent methods. However, the accuracy of each measurement was not sufficient to discriminate between an hexameric and an octameric structure of the enzyme oligomer. On the other hand, crosslinking experiments demonstrated the octameric structure of yeast phosphofructokinase. Obviously, some methods of molecular weight determination have led to erroneous results. In particular, our experiments show that the reliability of molecular weight determinations performed by gel filtration of native proteins must be considered with caution.