For almost twenty years, site-saturation mutagenesis (SSM) has been used to evolve stereoselective enzymes as catalysts for synthetic organic chemistry. Much of this work has focused on enzymes such as lipases and esterases, although the range is rapidly expanding. By contrast, using SSM to study enzyme mechanisms is much less common. Instead, site-directed mutagenesis is more generally employed, with a particular emphasis on alanine variants. In this review we provide examples of the growing use of SSM to study, not only substrate and reaction selectivity, but also the reaction mechanism of thiamin diphosphate (ThDP)-dependent enzymes. We report that using SSM to examine the roles of the catalytic residues of benzoylformate decarboxylase gave results that were at odds with earlier kinetic and structural studies using alanine substitutions, and brought their conclusions into question. SSM was also employed to examine the long held tenet that a bulky hydrophobic residue provides a fulcrum by which the V-conformation of the ThDP cofactor is maintained. X-ray structures showed that ThDP stayed in the V-conformation even when the replacement residues were charged, or did not contact the cofactor. We also summarize the results obtained when SSM was used to evolve new substrate specificity and/or enantioselectivity in ThDP-dependent enzymes such as benzoylformate decarboxylase (BFDC), transketolase, MenD, and the E1 component of the 2-oxoglutarate dehydrogenase complex. This article is protected by copyright. All rights reserved.