Galactose Mutarotase:  pH Dependence of Enzymatic Mutarotation

Here we report pH dependence of kinetic parameters for the mutarotation of α-d-glucose catalyzed by galactose mutarotase (GalM) from Escherichia coli. The values of k cat and k cat/K m for the mutarotation of α-d-galactose were found to be 1.84 × 104 s-1 and 4.6 × 106 M-1 s-1, respectively, at pH 7.0 and 27 °C. The corresponding values for α-d-glucose were 1.9 × 104 s-1 and 5.0 × 105 M-1 s-1. Inasmuch as the value of k cat/K m for the reaction of α-d-galactose is 10 times that for α-d-glucose, and the diffusional rate constants should be essentially the same for the two sugars, the mutarotation of α-d-glucose should not be diffusion controlled. Therefore, pH−rate profiles should not be distorted by diffusion. The k cat for the mutarotation of α-d-glucose is independent of pH. Therefore, either the enzyme−substrate complexes do not undergo ionization of catalytic groups, or the rate-limiting step is neither mutarotation nor diffusion. The profile of log k cat/K m versus pH is a distorted bell-shaped curve, with slopes of +1 on the acid side and −2 on the alkaline side. The values of pK a are 6.0 and 7.5, and mutarotation depends on the ionization states of three functional groups in the free enzyme, one unprotonated and two protonated. On the acid side, ring opening of α-d-glucose limits the rate, and on the alkaline side, ring closure of the open-chain sugar limits the rate. A mutarotation mechanism is presented in which one of the catalytic groups shuttles a proton to and from the endocyclic oxygen and the other two shuttle protons to the anomeric oxygen atoms. In this mechanism, three catalytic groups overcome the problem of nonstereospecificity in mutarotation. The groups are postulated to be His 104, His 175, and Glu 309. Mutations of these residues grossly impair catalytic activity. Variants H104Q- and E309Q-GalM display sufficient activity to allow profiles of log k cat/K m versus pH to be constructed. Both profiles show breaks on the acid side corresponding to pK a values of 5.8 for H104Q and 6.3 for E309Q. Apparently, ring opening of α-d-glucose limits the rate at low pHs, but ring closure does not become rate limiting at pHs up to 8.5 in reactions of these variants.