María Luz Cárdenas

Co-operativity in monomeric enzymes.

It has been known for at least 20 years that monomeric enzymes can in principle show kinetic behaviour similar in appearance to the binding of ligands to oligomeric proteins in which there are co-operative interactions between multiple binding sites. However, the initial lack of experimental examples of kinetic co-operativity suggested that in nature co-operativity always arose from interactions between binding sites. Now, however, several examples are known, most of which cannot be explained in terms of multiple binding sites on one polypeptide chain. All current theoretical models for monomeric co-operativity postulate that it arises from the presence in the mechanism of parallel pathways for substrate binding that are slow compared with the possible rate of the catalytic reaction. Rapid removal of the intermediates produced in the slow steps prevents them from approaching equilibrium and allows the appearance of kinetic properties that would not be possible in systems at equilibrium.

Understanding the parts in terms of the whole.

Abstract Metabolism is usually treated as a set of chemical reactions catalysed by separate enzymes. However, various complications, such as transport of molecules across membranes, physical association of different enzymes, giving the possibility of metabolite channelling, need to be taken into account. More generally, a proper understanding of the nature of life will require metabolism to be treated as a complete system, and not just as a collection of components. Certain properties of metabolic systems, such as feedback inhibition of the first committed step of a pathway, make sense only if one takes a broader view of a pathway than is usual in textbooks, so that one can appreciate ideas such as regulation of biosynthesis according to demand. More generally still, consideration of metabolism as a whole puts the emphasis on certain systemic aspects that are crucial but which can pass unnoticed if attention is always focussed on details. For example, a living organism, unlike any machine known or conceivable at present, makes and maintains itself and all of its components. Any serious investigation of how this can be possible implies an infinite regress in which each set of enzymes needed for the metabolic activity of the organism implies the existence of another set of enzymes to maintain them, which, in turn, implies another set, and so on indefinitely. Avoiding this implication of infinite regress represents a major challenge for future investigation.

Maintenance of the Monomeric Structure of Glucokinase under Reacting Conditions

Abstract Glucokinase is a monomeric protein under native and denaturating conditions yet presents a sigmoidal saturation function for glucose. These peculiarities suggested the possibility that polymerization occurs under assay conditions. Thus the apparent molecular weight of glucokinase was determined by gel filtration at 4 °C and at 30 °C in the presence of substrates and products, singly and in combination, creating during the filtration similar conditions as used in the assay. Gel filtration was performed also in the presence of N -acetylglucosamine, which is a competitive inhibitor and shifts to an hyperbole the saturation function for glucose. The same elution behavior, that is, a single symmetrical peak, was observed in every system used. This persistent monomeric form of glucokinase excludes the possibility that the sigmoidal function is the result of the interaction of different subunits. The possibility of an association-dissociation equilibrium in which the kinetic properties of the enzyme depend on the particular molecular weight species may also be rejected.

SPECIFICITY OF NON-MICHAELIS-MENTEN ENZYMES: NECESSARY INFORMATION FOR ANALYZING METABOLIC PATHWAYS

The specificity of an enzyme obeying the Michaelis−Menten equation is normally measured by comparing the kcat/Km for different substrates, but this is inappropriate for enzymes with a Hill coefficient h different from 1. The obvious alternative of generalizing Km in the expression as K0.5, the substrate concentration for half-saturation, is better, but it is not entirely satisfactory either, and here we show that kcat/K0.5(h) gives satisfactory results for analyzing the kinetic behavior of metabolic pathways. The importance of using kcat/K0.5(h) increases with the value of h, but even when h is small, it makes an appreciable difference, as illustrated for the mammalian hexokinases. Reinterpretation of data for the specificity of these enzymes in terms of the proposed definition indicates that hexokinase D, often believed highly specific for glucose, and accordingly called “glucokinase”, actually has the lowest preference for glucose over fructose of the four isoenzymes found in mammals.

Metabolic balance sheets.

Application of book-keeping principles to metabolic networks provides a powerful technique for understanding the properties of microorganisms and predicting the results of genetic modification.