The glmS ribozyme is a catalytic riboswitch that is activated for endonucleolytic cleavage by the coenzyme glucosamine-6-phosphate. Using kinetic assays and X-ray crystallography, we identify an active-site mutation of a conserved guanine that abolishes catalysis without perturbing coenzyme binding. Our results provide evidence that coenzyme function requires a specific nucleobase to interact with the nucleophile of the cleavage reaction.