The position-specific isotope effect of an enzymatic reaction is expressed as the ratio of rate constants for a monoisotopic substrate and a substrate substituted with one rare isotope. For example, enzyme formate dehydrogenase catalyzes the reaction of formate and NAD+ to carbon dioxide and NADH. The hydrogen of formate is directly transferred to NAD+. This step has an isotope effect, because the rate of protium transfer from formate to NAD+ is nearly three times faster than the rate of the same reaction with a deuterium transfer. This is also an example of a primary isotope effect. A primary isotope effect is one in which the rare isotope is substituted where a bond is broken or formed. Secondary isotope effects occur on other positions in the molecule and are controlled by the molecular geometry of the transition state. These are generally considered to be negligible but do arise in certain cases, especially for hydrogen isotopes.
Unlike abiotic reactions, enzymatic reactions occur through a series of steps, including substrate-enzyme binding, conversion of substrate to product, and dissociation of enzyme-product complex. The observed isotope effect of an enzyme will be controlled by the rate limiting step in this mechanism. If the step that converts substrate to product is rate limiting, the enzyme will express its intrinsic isotope effect, that of the bond forming or breaking reaction.