Substrate and metal ion binding to carbamate kinase: NMR and EPR studies |
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Authors: | RP Pillai M Marshall JJ Villafranca |
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Institution: | 1. Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 USA;2. Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706 USA |
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Abstract: | Metal ion and substrate binding to carbamate kinase from Streptococcus faecalis was studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance using Mn2+ as the paramagnetic probe. The enzyme binds Mn2+ weakly (KD = 0.45 ± 0.05 mm) with a stoichiometry of one per two subunits. However, in the presence of nucleotides, tighter binding of Mn2+ was observed with KD = 44 ± 4 μm in the presence of ADP and KD = 23 ± 4 μm with ATP present. Proton relaxation rate enhancement studies were conducted on water molecules interacting with ternary enzyme-Mn2+-nucleotide and binary enzyme-Mn2+ complexes. Mn2+ bound to carbamate kinase enhances the proton relaxation rate of water giving a binary enhancement value of ?b = 9.3 ± 0.4. When enzyme-Mn2+ was titrated with ADP or ATP, a bell-shaped titration curve was obtained typical of many other enzyme-Mn2+-nucleotide ternary complexes. Computer fits to the titration data gave ternary enhancement values of ?tADP = 14 ± 1 and ?tATP = 19 ± 1. The dissociation constants for Mn-ADP and Mn-ATP binding to carbamate kinase were also obtained from these data analyses and are K1 = 2.5 ± 0.5 μm and K1 = 50 ± 8 μm, respectively. Therefore, these data demonstrate the formation of a ternary enzyme-metal-nucleotide bridge complex at the nucleotide substrate site of carbamate kinase. Distance measurements were conducted by NMR techniques with 13C-enriched carbamate and demonstrate that carbamate is 4–8 Å from enzyme-bound Mn2+. Thus carbamate binds near the metal-nucleotide substrate site of carbamate kinase. |
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Keywords: | To whom to address correspondence at The Pennsylvania State University J J V is an Established Investigator of the American Heart Association |
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