A Search for a Solvent Isotope Effect on β‐Glucosidase

Solvent deuterium isotope effects can be a valuable tool in mechanistic enzymology to probe changes in hydrogen bonding interactions and general acid and base catalysis. We have examined the effect of substituting D 2 O for H 2 O on various equilibria and rates involving β‐glucosidase from sweet almond (EC 3.2.1.21 ). There is a normal isotope effect on the two pK a s (increase ~0.4 units in D 2 O) characterizing the pH/pD profile of k cat /K m , suggesting that the active site is not shielded from the solvent. However, this enzyme is unusual in that no significant solvent kinetic isotope effect (SKIE) is seen in either k cat or k cat /K m in reactions with substrates varying in reactivity by a factor of >6,000. For some of these substrates (e.g., methyl glucoside), the rate‐limiting step (rls) is formation of the glucosyl‐enzyme intermediate, while for others (e.g., 2,4‐dinitrophenyl glucoside) the rls is hydrolysis of the covalent intermediate. There is no significant SKIE observed with the poor substrate, methyl glucoside, or with the more efficient substrate, p ‐nitrophenyl glucoside. The lack of a detectable SKIE is surprising, since it is hard to imagine a mechanism for the highly efficient enzyme (catalytic proficiency ≈ 10 15 ) that does not involve acid/base catalysis in either of these steps. Also, there is no solvent equilibrium isotope effect (SEIE) on the binding of the substrate analog, p ‐nitrophenyl thioglucoside, nor on the binding of the product, or on a variety of competitive inhibitors, such as glucose. Although our search has been delayed by a hurricane, we are continuing to look for conditions (e.g., binding or reactivity of various substrates, affinity of various inhibitors) which might reveal a solvent isotope effect on almond β‐glucosidase.