Solvent isotope effects on a hydrolysis reaction catalysed by subtilisin and its N155G mutant. Failure of the proton inventory method to report hydrogen-bonding interactions in the oxyanion hole

Abstract

Proton inventories (solvent isotope effects in H₂O–D₂O mixtures) are not detectably different for the hydrolysis of an anilide substrate catalysed by subtilisin and its N155G mutant, despite the fact that this mutation removes a critical hydrogen-bonding interaction supplied by Asn-155 in the oxyanion hole of the enzyme, which stabilizes the transition state of this reaction by an amount equivalent to two orders of magnitude in reaction rate.