Characterization of the dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

The E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex (EC 1.2.4.1) is a thiamin diphosphate dependent enzyme that catalyzes oxidative decarboxylation of pyruvate. In the E1 component, two active center loops (an inner loop formed by residues 401-413, and outer loop formed by residues 541-557) become organized only on binding a intermediate analog which is capable of forming a stable thiamin diphosphate-bound covalent intermediate. In the ordered conformation many of the inner loop residues come into proximity of the active center and form a part of the active site channel. Kinetic, spectroscopic, and crystallographic studies on some inner loop residues and their variants led to the conclusion that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for (1) predecarboxylation events, (2) for sequestering active site chemistry from undesirable side reactions, (3) entry of ThDP and second molecule substrate and (4) for promoting communication between the E1 and E2 components of E. coli pyruvate dehydrogenase multienzyme complex.;Studies using site specific labeling (SSL) with thiol-directed probes sensitive to their local environment has immense potential to characterize the dynamics of these important regions. For this purpose five of six cysteine residues in parental E1 (120, 259, 575, 610, 654 and 770) were converted to alanines, the sixth C259 to asparagine, by site directed mutagenesis. This E1 variant without cysteines showed ∼4-fold reduction in activity compared to parental E1 in component-specific as well as overall complex activity assays. Moreover, reintroduction of cysteine for SSL studies in regions 1-55 (I11C) and 401-413 (Q408C) did not result in significant reduction in E1 activity retaining ∼40% and 80% overall activity, respectively, compared to the cysteineless E1 variant and ThDP or pre-decarboxylation steps. These results affirm that a cysteine-free construct creates a reliable system for characterization of E1 loop dynamics with the help of SSL studies.;Protein motions are ubiquitous and are intrinsically coupled to catalysis; their specific roles, however, remain largely elusive. Initial studies indicated that dynamic loops at the active center of the E1 component are essential for many of its catalytic functions starting from predecarboxylation steps to the transfer of the acetyl moiety to the E2 component. The kinetic resolution of pre-decarboxylation steps afforded by time-resolved circular dichroism studies revealed that formation of enzyme-ThDP-pyruvate Michaelis complex is extremely fast and could be diffusion limited. However, covalent addition of substrate to enzyme bound coenzyme (C-C bond formation) is found to be rate determining not only for pre-decaboxylation steps but also for overall E1 catalysis. Monitoring the steady state and time resolved kinetics of E1 and its loop variants at various solution viscosities revealed that kinetic steps, particularly C-C covalent bond formation, are indeed modulated by the loop dynamics. These observations taken together were strongly suggestive of an intimate correlation of catalysis and loop dynamics.;To determine the qualitative nature of these important and potentially correlated motions we used EPR spectroscopy, a highly sensitive method demonstrated in many studies to be exquisitely sensitive reporter of conformational changes. A cysteine-free E1 construct created for site-specific labeling with nitroxide on the inner loop revealed ligand induced conformational dynamics of the loop and a slow ‘open-close’ conformational equilibrium in the unliganded state. A 19F NMR label placed at various positions on the inner loop revealed motion on the ms to s time scale and led to a quantitative correlation of E1 catalysis and loop dynamics for the 200,000 Da protein. Thermodynamic studies showed that these motions coupled to catalysis reduce free energy of activation of covalent addition of substrate to the enzyme bound thiamin diphosphate by influencing both enthalpic and entropic components. This observation is significant considering the vast experimental data supporting electrostatic catalysis and differences surrounding the ‘dynamical hypothesis’ of enzyme catalysis. While data presented in this study do not argue against a generalized view favoring electrostatic catalysis in enzymes, they do place limitations on its predominance in E1. At the same time, the data strongly indicate ‘energetic harvesting of dynamics’ also play important role in catalysis as has been proposed by recent experimental and theoretical studies.;These results demonstrate efficient coupling of catalysis and regulation with enzyme dynamics and more importantly suggests a mechanism by which it may be achieved in a key branch-point enzyme in sugar metabolism reinforcing the hypothesis ascribing catalytic and regulatory roles to enzyme dynamics.