MECHANISM OF THE REACTION CATALYZED BY MANDELATE RACEMASE - STRUCTUREAND MECHANISTIC PROPERTIES OF THE D270N MUTANT

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro. J. A., Gerlt, J. A., Ko zarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J. , Fujita, J., & Petsko, G. A. (1994) Biochemistry. 33, 635-643], Lys 1 66 and His 297 are positioned appropriately to participate in catalysi s as acid/base catalysts, with Lys 166 participating as the (S)-specif ic acid/base catalyst and His 297 participating as the (R)-specific ac id/base catalyst. The dependence of k(cat) on pH for the racemization of both (R)- and (S)-mandelates suggests that the pK(a)s of the conjug ate acids of Lys 166 and His 297 are both similar to 6.4 [Landro, J. A ., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Ne idhart, D. J., & Kenyon, C. L. (1991) Biochemistry 30, 9274-9281; Kall arakal, A. T., Mitra, B., Kozarich, J. W., Gerlt, J. A., Clifton, J. R ., Petsko, G. A., Bi Kenyon, G. L. (1995) Biochemistry 34, 2788-2797]. Both acid/base catalysts are in close proximity to and approximately equidistant to the epsilon-ammonium group of Lys 164 and the essential Mg2+. The positive electrostatic potential provided by these cationic groups might be expected to increase the acidities of the cationic co njugate acids of the acid/base catalysts, thereby explaining the depre ssed pK(a) of Lys 166 but not the ''normal'' pK, of His 297. Asp 270 i s hydrogen bonded to N-delta of His 297 and, therefore, may allow the pK(a) of His 297 to be normal. In this paper we report the structural and mechanistic properties of the mutant in which Asp 270 is replaced with asparagine (D270N). The structure of D270N with (S)-atrolactate b ound in the active site reveals no geometric alterations in the active site when compared to the structure of wild-type MR complexed with (S )-atrolactate, with the exception that the side chain of His 297 is ti lted and displaced similar to 0.5 Angstrom away from Asn 270 and towar d the (S)-atrolactate. The k(cat)s for both (R)- and (S)-mandelates ar e reduced similar to 10(4)-fold. In accord with the proposal that Asp 270 influences the pK(a) of His 297, in the (R)- to (S)-direction no a scending limb is detected in the dependence of k(cat) on pH; instead, k(cat) decreases from a low pH plateau as described by a pK(a) of 10. In the (S)- to (R)-direction the dependence of k(cat) on pH is a bell- shaped curve that is described by pK(a)s of 6.4 and 10. In analogy to the previously reported properties of the H297N mutant [Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neid hart, D. J., & Kenyon, G. L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the alpha-proton of (S)- but no t (R)-mandelate with solvent and the stereospecific elimination of bro mide ion from (S)-p-(bromomethyl)mandelate. These observations suggest that His 297 and Asp 270 function as a catalytic dyad, with Asp 270 b eing at least partially responsible for the normal pK(a) of His 297 in wild-type MR.