311 Molecular and Cellular Biochemistry 184: 311–320, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands. Subtleties in control by metabolic channelling and enzyme organization Boris N. Kholodenko, 1,2 Johann M. Rohwer, 3 Marta Cascante 2 and Hans V. Westerhoff 3,4 1 A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia; 2 Universitat de Barcelona, Departmento de Bioquímica i Biología Molecular, Martí i Franquès 1, E–08028 Barcelona, Spain; 3 E.C. Slater Institute, BioCentrum, University of Amsterdam, Plantage Muidergracht 12, NL–1018 TV Amsterdam; 4 Department of Microbial Physiology, BioCentrum, Faculty of Biology, Free University, De Boelelaan 1087, NL–1081 HV Amsterdam, The Netherlands Abstract Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients. (Mol Cell Biochem 184: 311–320, 1998) Key words: metabolic channelling, non-ideal metabolism, control coefficient, enzyme-enzyme interactions, macromolecular crowding, bacterial phosphotransferase system Introduction In a standard view cellular metabolism is presented in terms of so-called ‘ideal’ metabolic pathways. This paradigm implies that the catalysts (enzymes) interact only through bulk-phase intermediates in well defined compartments containing well- mixed metabolite pools. However, it has long been recognized by biochemists that aspects of cellular metabolism are or- ganized both structurally and temporarily (see [1, 2] for recent reviews). Organized (non-ideal) metabolism comes in many forms, e.g., direct transfer of intermediates (‘channelling’), dynamic versus static channelling [3, 4], restricted diffusion [5], local coupling [6] and macromolecular crowding [7, 8]. Recently developed extensions of metabolic control analysis (reviewed in [9]) make it possible to predict and analyse the implications of various types of enzyme organization for the regulation and control of cell function. Enzyme-enzyme interactions can result in many special control properties. In this chapter we shall review the subtleties of the control brought about by metabolic channelling and Present address: B.N. Kholodenko, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, JAH, 1020 Locust Street, Philadelphia, PA 19107, USA Present address: J.M. Rohwer, Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa Address for offprints: B.N. Kholodenko, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, JAH, 1020 Locust Street, Philadelphia, PA 19107, USA