JOURNAL OF MOLECULAR RECOGNITION, VOL. zyxwvut 6, 159-165 (1993) zyxwvu Enzymology zyxw In Viuo using NMR and Molecular Genetics Kevin M. Brindle,* Alexandra M. Fulton and Simon-Peter Williams Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 IQW, UK Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured zyxwvutsr in uitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substrate and effector concentrations and possible interactions with other cellular macromolecules, which may modify their kinetic properties. There is a considerable incentive, therefore, to study the properties of enzymes directly in uiuo. We have been using non- invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in uiuo. We have also developed a novel labelling strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measure the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in uiuo INTRODUCTION An undergraduate reader of a basic biochemistry text book could be forgiven for coming away with the idea that the controls of the central pathways of cellular metabolism are thoroughly understood. This mislead- ing impression is created, in part, by the detailed molecular description that we have of the components of these pathways. For some pathways the genes for the component enzymes have been cloned and sequenced and in many cases there are high resolution x-ray crystal structures. This is the case for the glycolytic pathway for example (Fothergill-Gilmore and Michels, 1993). This is the triumph of reductionism, the break- ing down of the cell into its component parts followed by detailed studies of the structure and function of the individual components. However, while this approach underpins modern biochemistry and has undoubtedly been of enormous value, the information obtained does not necessarily tell us how these isolated components work and interact in zyxwvutsrqp uiuo, i.e., in the intact cell. Kell and Welch (1991), when describing this problem, evoke the tale of Humpty Dumpty. In the words of the nursery rhyme: ‘all the King’s horses and all the King’s men couldn’t put Humpty (the cell) together again’. In breaking the cell open to study its component parts vital information is lost regarding their organization. Rees, in a recent address to the Royal Society (Rees, 1993), envisaged a post-reductionist era in biochemistry in which the new challenge will be to understand how the protein machinery of cells works in uiuo. He sug- gested that we will need new ways of looking inside cells; “opportunities will multiply for new methods of non-destructive observation and measurements on molecular and cellular events in intact tissues zyxwvut . . . The technologies of the future will give us precise quantita- Author to whom correspondence should be addressed Abbreviations used: PFKl , zyxwvut 6-phosphofructo-I-kinase; MCA, meta- bolic control analysis; PCr, phosphocreatine; GAPDH, glyceraldehyde-%phosphate dehyrogenase; PGK, phosphoglycerate kinase. tive measurements on, for example, molecular changes in local sites in the living organism”. We have been using non-invasive NMR techniques, in conjunction with molecular genetic methods for altering enzyme levels and their properties, to investi- gate the kinetic and physical properties of specific enzymes in the intact cell (Brindle, 1988a; Brindle et al., 1989, 1990, 1991; Davies and Brindle, 1992; Williams et al., 1993). We have used as a model system the yeast Saccharomyces cereuisiae since it is easy to grow in the large (gram) quantities needed for in uiuo NMR experiments and the techniques for the genetic manipulation of this organism are well established. The metabolic pathways that we have chosen for examin- ation are two which are fundamental to cellular energy generation and as a consequence have been very well studied using classical biochemical techniques; glycoly- sis and mitochondria1 oxidative phosphorylation. CONTROL OF GLYCOLYTIC FLUX The conventional view of glycolysis is that it is controlled by three enzymes which catalyse reactions that are effectively irreversible in the cell, i.e., hexoki- nase, 6-phosphofructo-1-kinase (PFK1) and pyruvate kinase. PFK is usually thought to be the primary controlling enzyme. For example Stryer, in his well known text book (Stryer, 1988), refers to PFKl in the following way: “Phosphofructokinase, which catalyses the committed step in glycolysis, is the most important control site”. However this rather qualitative view of metabolic control has been challenged in recent years by the quantitative rigour of metabolic control analysis (MCA) . Metabolic control analysis MCA arose out of the landmark papers by Kacser and Burns (1973) and Heinrich and Rapoport (1974). These workers introduced a relatively simple theoretical CCC 0952-349~/~3/040159-07 zyxwvutsrq 0 1993 by John Wiley & Sons, Ltd Accepted 21 September 1993