Mandelate racemase. V. Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis

摘要: The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit a dissociation constant 8 muM, in agreement its kinetically determined activator constant. Also, six additional ions the enzyme, much more weakly, 1.5 mM. Binding at tight site enhances effect on longitudinal relaxation rate (1/T1p) water protons factor 11.9 24.3 MHz. From frequency dependence 1/T1p, it that there are similar 3 ligands enzyme-bound which exchange larger than or equal 10-7 sec-1. correlation time for Mn2+-water interaction is frequency-dependent, indicating be dominated spin Mn2+. Formation ternary enzyme-Mn2+-mandelate complex decreases number fast exchanging 1, but does not affect tau-c, suggesting displacement occlusion ligand. D,L-alpha-phenylglycerate salicylate produce little no change enzyme-Mn2+-H2O interaction, complexes detected indirectly changes enzyme-Mn2+ mutual competition experiments. In all cases constants substrates from magnetic titrations agree K-M K-i values therefore reflect active complexes. effects 1/T1 1/T2 13C-enriched carbons 1-[13C]-D,L-mandelate 2-[13C]-D,L-mandelate, carboxylate carbon carbinol distances 2.93 plus minus 0.04 2.71 A, respectively, calculated, bidentate chelation binary Mn2+-mandelate complex. Mn2+, D,L-mandelate, these increase 5.5 0.2 7.2 presence least 98.9% second sphere C1 C2 atoms linear array. data suggest ligand immobilized between bound substrate. This intervening may polarize protonate carboxyl group. 1/T2p substrate this (larger 5.6 times 10-4 sec-1) 52 greater maximal turnover (1070 sec-1), nuclear (NMR) competent participate catalysis. Relationships among microscopic considered.