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

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• 作者：Susan L. Schafer ; William C. Barrett ; Abraham T. Kallarakal ; Bharati Mitra ; John W. Kozarich ; John A. Gerlt ; James G. Clifton ; Gregory A. Petsko ; and George L. Kenyon
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：May 7, 1996
• 年：1996
• 卷：35
• 期：18
• 页码：5662 - 5669
• 全文大小：433K
• 年卷期：v.35,no.18(May 7, 1996)
• ISSN：1520-4995

文摘

On the basis of the available high-resolutionstructures of mandelate racemase (MR) fromPseudomonas 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 His297are positioned appropriately to participate in catalysis as acid/basecatalysts, with Lys 166 participatingas the (S)-specific acid/base catalyst and His 297participating as the (R)-specific acid/base catalyst.Thedependence of k_cat on pH for the racemization ofboth (R)- and (S)-mandelates suggests that thepK_as ofthe 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., & 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/basecatalysts are in close proximity to and approximatelyequidistant to the -ammonium group of Lys 164 and the essentialMg²⁺. The positive electrostaticpotentialprovided by these cationic groups might be expected to increase theacidities of the cationic conjugateacids of the acid/base catalysts, thereby explaining the depressedpK_a of Lys 166 but not the"normal"pK_a of His 297. Asp 270 is hydrogen bondedto N of His 297 and, therefore, may allow thepK_a of His297 to be normal. In this paper we report the structural andmechanistic properties of the mutant inwhich Asp 270 is replaced with asparagine (D270N). The structureof D270N with (S)-atrolactate boundin the active site reveals no geometric alterations in the active sitewhen compared to the structure ofwild-type MR complexed with (S)-atrolactate, with theexception that the side chain of His 297 is tiltedand displaced ~0.5 Å away from Asn 270 and toward the(S)-atrolactate. The k_cats forboth (R)- and(S)-mandelates are reduced ~10⁴-fold. Inaccord with the proposal that Asp 270 influences thepK_a ofHis 297, in the (R)- to (S)-direction noascending limb is detected in the dependence ofk_cat on pH; instead,k_cat decreases from a low pH plateau asdescribed by a pK_a of 10. In the(S)- to (R)-direction the dependenceof k_cat on pH is a bell-shaped curve that isdescribed by pK_as of 6.4 and 10. In analogyto the previouslyreported 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 catalyzesboth the facile exchange of the -proton of (S)- but not(R)-mandelate with solvent and thestereospecificelimination of bromide ion from(S)-p-(bromomethyl)mandelate. Theseobservations suggest that His297 and Asp 270 function as a catalytic dyad, with Asp 270 being atleast partially responsible for thenormal pK_a of His 297 in wild-typeMR.