title: Energy changes during the formation and interconversion of enzyme-substrate complexes.
abstract: The rate constants and equilibrium constants of the individual steps of several enzyme reactions may be determined by the application of rapid reaction methods and isotope techniques. This makes it possible to complement the formalism of the Haldane relation with details of the reaction mechanism. It has been shown that, in several enzyme reactions, steps involving chemical catalysis are fast and have small free-energy changes compared with those of the substrate binding and product dissociation processes. Data are presented in this paper for three enzyme reactions for which different methods have been used to elucidate the kinetic parameters of the elementary steps. For cardiac lactate dehydrogenase (EC 1.1.1.27), absorption and fluorescence spectroscopy have been used to distinguish the step involved in the chemical process from those involved in the formation of the substrate complex and the release of the product. The rate of interconversion between enzyme-bound substrates and products is fast compared with other steps and the equilibrium constant for the process is near unity. Consequently, the difference of standard free energy changes for the formation of the two ternary complexes correspons approximately to the overall free-energy change of the hydrogen transfer reaction. Isotope kinetic techniques can be used to study the reactions of triosephosphate isomerase (EC 5.3.1.1). With this enzyme, the interconversion of enzyme-bound substrate into product is comparable in rate to product dissociation. The reactions of myosin subfragment 1 with ATP, studied by fluorescence spectroscopy and chemical quenching, follow a similar pattern in that the equilibrium constant of the chemical step in which water reacts with protein-bound ATP is 9. In this case, however, there is a remarkably large free-energy change associated with a first-order process involved in the binding of ATP. The possible significance of these results to energy transduction in muscle contraction as well as in the biosynthesis of ATP is discussed.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002384:Catalysis; D004735:Energy Transfer; D004798:Enzymes / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008722:Methods; D009218:Myosins / Q000378:metabolism; D010103:Oxygen Isotopes; D013050:Spectrometry, Fluorescence; D013552:Swine; D013696:Temperature; D013816:Thermodynamics; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*; D014867:Water / Q000378:metabolism
chemical_list: D004798:Enzymes; D010103:Oxygen Isotopes; D014867:Water; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D000251:Adenosine Triphosphatases; D009218:Myosins; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase