The biotin holoenzyme synthetases (BHS) are essential enzymes in all organisms that catalyze post-translational linkage of biotin to biotin-dependent carboxylases. The primary sequences of a large number of these enzymes are now available and homologies are found among all. The glycine-rich sequence, GRGRXG, constitutes one of the homologous regions in these enzymes and, based on its similarity to sequences found in a number of mononucleotide binding enzymes, has been proposed to function in ATP binding in the BHSs. In the Escherichia coli enzyme, the only member of the family for which a three-dimensional structure has been determined, the conserved sequence is found in a partially disordered surface loop. Mutations in the sequence have previously been isolated and characterized in vivo. In this work these single-site mutants, G115S, R118G, and R119W, of the E. coli BHS have been purified and biochemically characterized with respect to binding of small molecule substrates and the intermediate in the biotinylation reaction. Results of this characterization indicate that, rather than functioning in ATP binding, this glycine-rich sequence is required for binding the substrate biotin and the intermediate in the biotinylation reaction, biotinyl-5′-AMP. These results are of general significance for understanding structure-function relationships in biotin holoenzyme synthetases.