Archives of Biochemistry and Biophysics 2013-08-01

Kinetic mechanism and energetics of binding of phosphoryl group acceptors to Mycobacterium tuberculosis cytidine monophosphate kinase.

Léia Jaskulski, Leonardo A Rosado, Diana C Rostirolla, Luis F S M Timmers, Osmar N de Souza, Diogenes S Santos, Luiz A Basso

Index: Arch. Biochem. Biophys. 536(1) , 53-63, (2013)

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Abstract

Cytidine monophosphate kinase from Mycobacterium tuberculosis (MtCMK) likely plays a role in supplying precursors for nucleic acid synthesis. MtCMK catalyzes the ATP-dependent phosphoryl group transfer preferentially to CMP and dCMP. Initial velocity studies and Isothermal titration calorimetry (ITC) measurements showed that MtCMK follows a random-order mechanism of substrate (CMP and ATP) binding, and an ordered mechanism for product release, in which ADP is released first followed by CDP. The thermodynamic signatures of CMP and CDP binding to MtCMK showed favorable enthalpy and unfavorable entropy, and ATP binding was characterized by favorable changes in enthalpy and entropy. The contribution of linked protonation events to the energetics of MtCMK:phosphoryl group acceptor binary complex formation suggested a net gain of protons. Values for the pKa of a likely chemical group involved in proton exchange and for the intrinsic binding enthalpy were calculated. The Asp187 side chain of MtCMK is suggested as the likely candidate for the protonation event. Data on thermodynamics of binary complex formation were collected to evaluate the contribution of 2'-OH group to intermolecular interactions. The data are discussed in light of functional and structural comparisons between CMP/dCMP kinases and UMP/CMP ones. Copyright © 2013 Elsevier Inc. All rights reserved.