Binding energy and catalysis: deoxyfluoro sugars as probes of hydrogen bonding in phosphoglucomutase.

Estimates of the contributions of hydrogen-bonding interactions with each of the sugar hydroxyls to the binding of the substrate alpha-D-glucopyranosyl phosphate both in the ground state and at the transition state for the initial phosphoryl transfer have been obtained by kinetic studies. Michaelis parameters (kcat and Km) for a complete series of deoxy- and deoxyfluoro-alpha-D-glucopyranosyl phosphates provide insight into specific interactions with each hydroxyl at the transition state. Inhibition constants (Ki) for a series of deoxygenated and fluorinated analogues of the competitive inhibitor 6-deoxy-6-fluoro-alpha-D-glucopyranosyl phosphate provide insight into ground-state interactions. Interactions at each hydroxyl are found to strengthen only slightly upon progressing from the ground state to the transition state in contrast to that seen with glycogen phosphorylase [Street et al. (1989) Biochemistry 28, 1581] where transition-state interactions became much stronger. This is in accord with the mechanisms for these two enzymes where no distortion of the sugar ring occurs for phosphoglucomutase, whereas considerable distortion is expected for glycogen phosphorylase.