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Journal of Bacteriology, February 2006, p. 1143-1154, Vol. 188, No. 3
0021-9193/06/$08.00+0     doi:10.1128/JB.188.3.1143-1154.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from Methanosarcina thermophila

Sarah H. Lawrence,^1, Kelvin B. Luther,² Hermann Schindelin,^2* and James G. Ferry^1*

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500,¹ Department of Biochemistry, Center for Structural Biology, SUNY Stony Brook, Stony Brook, New York 11794-5115²

Received 15 August 2005/ Accepted 18 October 2005

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila in complex with the substrate CoA revealed one CoA (CoA¹) bound in the proposed active site cleft and an additional CoA (CoA²) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [K_D], 20 µM) and low-affinity (K_D, 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA¹ is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA² and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser³⁰⁹ and Arg³¹⁰, which are proximal to the reactive sulfhydryl of CoA¹. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp³¹⁶, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg³¹⁰ binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser³⁰⁹.

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* Corresponding author. Mailing address for Hermann Schindelin: Department of Biochemistry, Center for Structural Biology, SUNY Stony Brook, Stony Brook, NY 11794-5115. Phone: (631) 632-1022. Fax: (631) 632-1555. E-mail: hermann.schindelin{at}sunysb.edu. Mailing address for James G. Ferry: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802-4500. Phone: (814) 863-5721. Fax: (814) 863-6217. E-mail: jgf3{at}psu.edu.

Present address: Fox Chase Cancer Center, Philadelphia, PA 19111-2497.

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Journal of Bacteriology, February 2006, p. 1143-1154, Vol. 188, No. 3
0021-9193/06/$08.00+0     doi:10.1128/JB.188.3.1143-1154.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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