Identification of the Escherichia coli enzyme I binding site in histidine-containing protein, HPr, by the effects of mutagenesis

The structure of the N-terminal domain of enzyme I complexed with histidine -containing protein (HPr) has been described by multi-dimensional NMR. Resi dues in HPr involved in binding were identified by intermolecular nuclear O verhauser effects (Garrett et al. 1999). Most of these residues have been m utated, and the effect of these changes on binding has been assessed by enz yme I kinetic measurement. Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Le u47, Lys49, Gln51, and Thr56 result in increases to the HPr K-m of enzyme I , which would be compatible with changes in binding. Except for mutations t o His15 and Arg17, very little or no change in V-max was found. Alanine rep lacements for Gln21, Thr52, and Leu55 have no effect. The mutation Lys40Ala also affects HPr K-m of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR. The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala , Lys27Ala, and Lys49Gly) caused relatively large increases in K-m (>5-fold ) indicating these residues have more significant roles in binding to enzym e I. Acidic replacement at Ser46 caused very large increases (>100-fold), w hile Gln51Glu gave a 3-fold increase in K-m. While these results essentiall y concur with the identification of residues by the NMR experiments, the ap parent importance of individual residues as determined by mutation and kine tic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects.