Mechanism of the Reaction Catalyzed by Mandelate Racemase:  Structure and Mechanistic Properties of the D270N Mutant^,

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., & 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 kcat on pH for the racemization of both (R)- and (S)-mandelates suggests that the pKaS of the conjugate acids of Lys 166 and His 297 are both approximately 6.4 [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., 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., & 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 conjugate acids of the acid/base catalysts, thereby explaining the depressed pKa of Lys 166 but not the "normal" pKa of His 297. Asp 270 is hydrogen bonded of N delta of His 297 and, therefore, may allow the pKa 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 approximately 0.5 A away from Asn 270 and toward the (S)-atrolactate. The kcatS for both (R)- and (S)-mandelates are reduced approximately 10(4)-fold. In accord with the proposal that Asp 270 influences the pKa of His 297, in the (R)- to (S)-direction no ascending limb is detected in the dependence of kcat of pH; instead, kcat decreases from a low pH plateau as described by a pKa of 10. In the (S)- to (R)-direction the dependence of kcat of pH is a bell-shaped curve that is described by pKaS 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., & Kenyon, G.L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the alpha-proton of (S)- but not (R)-mandelate with solvent and the stereospecific elimination of bromide ion from (S)-p-(bromomethyl)mandalate. 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 pKa of His 297 in wild-type MR.

Crystal Structure of dTDP-4-keto-6-deoxy-d-hexulose 3,5-Epimerase fromMethanobacterium thermoautotrophicum Complexed with dTDP

“…Interestingly, the ⑀-imine of His-64 is hydrogen-bonded to one of the carboxylates of Asp-172 and similarly for His-120 with Asp-84. Interactions between His and Asp residues of this nature were observed in the active site of mandelate racemase (MR) where they functioned as catalytic dyads in the acid/base mechanism (12). There are also a number of well ordered water molecules occupying this region of the active site and they are within hydrogen-bonding distance to the hexulose moiety of the substrate.…”

Investigation of the Roles of Catalytic Residues in Serotonin N-Acetyltransferase

“…However, the H122Q mutation resulted in only a 6-fold drop in the chemical step, a reduction well below the maximal effect of general base residue contributions in other enzyme reactions, where 10,000-fold rate effects upon deletion of general base residues are not uncommon (22,23). It was a formal possibility that the nearby residue His-120 could function as the catalytic base, "rescuing" the phenotype of the mutant.…”

Glutamate 170 of Human l-3-Hydroxyacyl-CoA Dehydrogenase Is Required for Proper Orientation of the Catalytic Histidine and Structural Integrity of the Enzyme

“…As discussed by Levy and co-workers (15), the aspartate residue of such active site pairs has been postulated to serve various functions. In the case of glucose 6-phosphate dehydrogenase (15) and mandelate racemase (14), it appears that the aspartate residue facilitates catalysis by altering the pK a of the histidine. In both cases, the position of the histidine residue is relatively undisturbed by replacement of aspartate with an asparagine residue.…”