Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4- epimerase from Escherichia coli

Synthesis and overexpression of a gene encoding Escherichia coil UDP- galactose 4-epimerase and engineered to facilitate cassette mutagenesis are described. General acid-base catalysis at the active site of this epimerase has been studied by kinetic and spectroscopic analysis of the wild-type enzyme and its specifically mutated forms Y149F, S124A, S124V, and S124T. The X-ray crystal structure of Y149F as its abortive complex with UDP-glucose is structurally similar to that of the corresponding wild-type complex, except for the absence of the phenolic oxygen of Tyr 149. The major effects of mutations are expressed in the values of k(cat) and k(cat)/K(m). The least active mutant is Y149F, for which the value of k(cat) is 0.010% of that of the wild-type epimerase. The activity of S124A is also very low, with a k(cat) value that is 0.035% of that of the native enzyme. The values of K(m) for Y149F and S124A are 12 and 21% of that of the wild-type enzyme, respectively. The value of k(cat) for S124T is about 30% of that of the wild- type enzyme, and the value of K(m) is similar to that of the native enzyme. The reactivities of the mutants in UMP-dependent reductive inactivation by glucose are similarly affected, with kobs being decreased by 6560-, 370-, and 3.4-fold for Y149F, S124A, and S124T, respectively. The second-order rate constants for reductive inactivation by NaBH₃CN, which does not require general base catalysis, are similar to that for the native enzyme in the cases of S124A, S124T, and S124V. However, Y149F reacts with NaBH₃CN 12-20- fold faster than the wild-type enzyme at pH 8.5 and 7.0, respectively. The increased rate for Y149F is attributed to the weakened charge-transfer interaction between Phe 149 and NAD⁺, which is present with Tyr 149 in the wild-type enzyme. The charge-transfer band is present in the serine mutants, and its intensity at 320 nm is pH-dependent. The pH dependencies of A₃₂₀ showed that the pK(a) values for Tyr 149 are 6.08 for the wild-type epimerase, 6.71 for S124A, 6.86 for S124V, and 6.28 for S124T. The low pK(a) value for Tyr 149 is attributed mainly to the positive electrostatic field created by NAD⁺ and Lys 153 (4.5 kcal mol^-1) and partly to hydrogen bonding with Ser 124 (1 kcal mol^-1). The pK(a) of Tyr 149 is the same as the kinetic pK(a) for the Bronsted base that facilitates hydride transfer to NAD⁺. We concluded that Tyr 149 provides the driving force for general acid- base catalysis, with Ser 124 playing an important role in mediating proton transfer.