Metabolic Distress Associated with Impaired Control by Alternative Substrates: Two Examples taken from Purine Metabolism

Abstract

Metabolic control analysis has developed from the conviction that ordinary kinetic analysis of enzymes that have been separated from their physiological neighbours cannot allow any prediction of the metabolic flux through the pathway in which the enzymes are embedded in vivo(Cornish-Bowden, 1995). Accordingly, steady-state kinetics have been regarded as a useful tool for understanding the molecular mechanisms of catalysis as well as the relationship between structure and function in the enzymatic proteins, but nothing else. For instance, the ping-pong mechanism in aspartate aminotransferase can be related with the presence of a single substrate-binding site on the catalytic subunit (Segel, 1975), while the pattern for transcarboxylase, which possesses two separate and independent sites, is conceivably consistent with hybrid ping-pong/rapid-equilibrium-random kinetics (Northrop, 1969). Enzymes lacking coenzymes usually require the formation of ternary complexes. Ordered sequential mechanisms have been attributed to steric hindrance of substrates and/or to changes in protein conformation during catalysis. Extensive literature on specific examples have been the object of several reviews (Cleland, 1967; Segel, 1975; Cornish-Bowden, 1995). Here, we point out that the steady-state kinetic analysis, despite being generally regarded as a solved problem, is far from trivial and might give new insight for understanding the role of the enzymes in their physiological context. We will focus our attention on enzymes with broad specificity that can use alternative substrates in different metabolic pathways.