The decrease in the rate of bond cleavage resulting from the presence of heavy isotopes is expressed to a greater or lesser extent as an isotope effect on steady-state kinetic parameters of enzyme-catalyzed reactions, depending upon complex relationships between individual rate constants. This paper describes these relationships and derives general kinetic expressions which allow the determination of the limits of the relative contribution of different reaction components to Kmax and Hmax/Wm. The value of the true isotope effect on a carbon-hydrogen bond breaking step, I. he major reason for determining kinetic isotope effects in enzymology has been to determine whether the maximal velocity is a measure of the rate of the step in which covalent change takes place (Jencks, 1969). Such a step is normally identified as the "rate-determining step" of the enzymatic reaction. The results to date and the steady-state concepts of current kinetic theory suggest, however, that the domination of maximal velocities of enzymatic reactions by a single covalent step is a rare event. It now appears likely that the maximal velocity of most enzymes is dependent upon several "rate-contributing" or "partially rate-limiting" steps. In this latter situation, apparent isotope effects have meaning only in terms of a comparison to the true isotope effects exerted on the catalytic step of covalent change.However, since true isotope effects could not previously be determined, the usual procedure has been to evaluate apparent effects on an absolute scale, based upon an implied comparison with apparent effects for other enzymes and results obtained in chemical reactions. Such an approach ignores the possible wide variation of the true isotope effects in enzyme-catalyzed reactions, which are, in addition, probably much greater than the current standards of comparison.

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Seven proton transfers in five steps participate in a catalytic turnover of an aspartic protease. The Rosetta Stone for elucidating their role is a low-barrier hydrogen bond that holds the two aspartic carboxyls in a coplanar conformation. The proton of this bond shuttles between oxygens during chemical steps via hydrogen tunneling, unlike in previous proposals where it was transferred to substrate. After the release of products, both carboxyls are protonated and the bond is missing. Re-forming the bond is a significant step within a kinetic isomechanism. The bond also explains-at long last-the extremely low pK in pH profiles.

Most biochemistry textbooks describe V/K, or k cat/K m, as one of the fundamental kinetic constants for catalysis in enzymatic reactions and associate it with some measure of the rate of the chemical transformation of substrate into product. However, in the reactions of all enzymes except isomerases and mutases, V/K fails to encompass a complete turnover. Instead, it can be shown that V/K actually provides a measure of the rate of capture of substrate by free enzyme into a productive complex or complexes destined to form products and complete a turnover at some later time. Similarly, V or k cat provides a measure of the rate of release of product from the productive enzyme complexes that constitute capture. It is here suggested that the symbols V/K and k cat be replaced by k cap and k rel, respectively, at least in the teaching of enzyme kinetics. Capture and release are equally necessary to generate a complete catalytic turnover, but they are determined by different things, and the proposed symbolism is less abstract than older alternatives. Used together, they provide a more accurate definition of the Michaelis constant, as K m = k rel/k cap, which is the kinetic equivalent of the thermodynamic dissociation constant, K d = k off /k on.

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