We have employed site-directed mutagenesis to investigate the contribution of a conserved arginyl residue to the catalytic activity and cofactor affinity of D-serine dehydratase, a model pyridoxal 5′-phosphate (vitamin B6) enzyme. Replacement of R-120 in the active site peptide of D-serine dehydratase by L decreased the affinity of the enzyme for pyridoxal 5′-phosphate by 20-fold and reduced turnover by 5–8-fold. kcatdisplayed modified substrate α-deuterium isotope effects and altered dependence on both temperature and pH. Analysis of the pH rate profiles of DSD and the R-120 → L variant indicated that R-120 interacts electrostatically with catalytically essential ionizable groups at the active site of wild type D-serine dehydratase. The decrease in cofactor affinity observed for DSD(R120L) was not accompanied by significant perturbations in the UV, CD, or 31P NMR spectrum of the holoenzyme, suggesting that the contribution of R-120 to pyridoxal phosphate affinity may be indirect or else involve an interaction with a cofactor functional group other than the 5′-phosphoryl moiety.