Steady-State Enzyme Kinetics at High Pressure

Pressure effects on enzyme-catalyzed reactions were traditionally interpreted within a simplistic kinetic mechanism in which the transition state presented the highest energy barrier, and the chemical transformation of substrate to product was considered to be a singular, rate-limiting step. This was also true of isotope effects on enzyme-catalyzed reactions, but extensive isotopic studies have led to the conclusion that this transition state is rarely the highest barrier. Rather, the release of products (or the conformational change preceding product release) is usually the slowest step, often accompanied by several other partially ratelimiting steps. Thus, our interpretations of pressure effects must be shifted accordingly. Values attributed to AV‡ have been determined for more than 50 enzymes, more of them with a positive sign than negative, and most in the range of 20 to 40 ml mol–1 but these may or may not have anything to do with the activation volume associated with the transition state. Volume changes specifically associated with the binding of ligands to enzymes have been reported as well, including some very large values, as high as ΔV = 85 ml mo–1. Kinetically, these equilibrium pressure effects also originate in conformational changes because water is not very compressible; hence, rates of diffusion to and from enzymes are virtually unaffected by pressure. Much larger changes, as high as ΔV = –391 mi mo–1, have been observed during disaggregation and denaturation of enzymes. Thus, while it is possible for pressure effects to be expressed on every step of an enzymatic reaction, and to cause denaturation as well, making kinetic data from pressure effects hopelessly complex and uninterpretable, it appears likely that the most significant pressure effects will be expressed on conformational changes associated with product dissociations, without much kinetic complexity. This makes sense from another point of view—that the largest volume changes are probably on solvation equilibria during ligand binding and protein folding. Pressure effects on isotope effects have the potential of specifically identifying whether or not a volume change occurs upon attaining the transition state. With the exception of hydrogen tunneling, intrinsic isotope effects are independent of pressure.