tailieunhanh - Báo cáo khoa học: The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme–substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine sub-strate in the enzyme–substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the sug-gestion that it is the protonated form of the amine substrate that binds to the enzyme. | ễFEBS Journal The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex Rachel V. Dunn1 Ker R. Marshall2 Andrew W. Munro1 and Nigel S. Scrutton1 1 Faculty of Life Sciences Manchester Interdisciplinary Biocentre University of Manchester UK 2 Department of Biochemistry University of Leicester UK Keywords kinetic isotope effect mechanism monoamine oxidase pH dependence Correspondence N. S. Scrutton Faculty of Life Sciences Manchester Interdisciplinary Biocentre University of Manchester 131 Princess Street Manchester M1 7DN UK Fax 44 161 3068918 Tel 44 161 3065152 E-mail Received 10 April2008 revised 25 May 2008 accepted 2 June 2008 doi A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pKa of . In stopped-flow assays the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant kred Ks reporting on ionizations in the free enzyme and or free substrate is due to deprotonation of the free substrate and the .

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