Mechanism of the reaction catalyzed by mandelate racemase: Structure and mechanistic properties of the D270N mutant

Susan L. Schafer, William C. Barrett, Abraham T. Kallarakal, Bharati Mitra, John W. Kozarich, John A. Gerlt, James G. Clifton, Gregory A. Petsko, George L. Kenyon

Research output: Contribution to journalArticlepeer-review

Abstract

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., and Petsko, G. A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/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 conjugate acids of Lys 166 and His 297 are both ~6.4 [Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., and Kenyon, G. L. (1991) Biochemistry 30, 9274-9281; Kallarakal, A. T., Mitra, B., Kozarich, J. W., Gerlt, J. A., Clifton, J. R., Petsko, G. A., and Kenyon, G. L. (1995) Biochemistry 34, 2788-2797]. Both acid/base catalysts are in close proximity to and approximately equidistant to the ε-ammonium group of Lys 164 and the essential Mg20. The positive electrostatic potential provided by these cationic groups might be expected to increase the acidities of the cationic conjugate acids of the acid/base catalysts, thereby explaining the depressed pK(a) of Lys 166 but not the 'normal' pK(a) of His 297. Asp 270 is hydrogen bonded to N(δ) 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 bound 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 tilted and displaced ~0.5 Å away from Asn 270 and toward the (S)-atrolactate. The k(cat)s for both (R)- and (S)-mandelates are reduced ~104-fold. In accord with the proposal that Asp 270 influences the pK(a) of His 297, in the (R)- to (S)-direction no ascending 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., Neidhart, D. J., and Kenyon, G. L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the α-proton of (S)- but not (R)-mandelate with solvent and the stereospecific elimination of bromide ion from (S)-p- (bromomethyl)mandelate. These observations suggest that His 297 and Asp 270 function as a catalytic dyad, with Asp 270 being at least partially responsible for the normal pK(a) of His 297 in wild-type MR.

Original languageEnglish (US)
Pages (from-to)5662-5669
Number of pages8
JournalBiochemistry
Volume35
Issue number18
DOIs
StatePublished - May 7 1996

ASJC Scopus subject areas

  • Biochemistry

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