DIRECT MEASUREMENTS OF THE DISSOCIATION-RATE CONSTANT FOR INHIBITOR-ENZYME COMPLEXES VIA THE T-1-RHO AND T-2 (CPMG) METHODS

The unimolecular dissociation rate constant, k(-1), for the inhibitor-enzyme complex tubercidin-Escherichia coli purine nucleoside phosphorylase (PNPase) has been determined directly via two related H-1 NMR methods for studying exchange-mediated transverse relaxation. One method involves measurements of the decay rate, 1/T-1 rho, of spin-locked magnetization in the rotating frame as a function of the strength of the spin-locking field, omega(SL) The second method involves measurements of the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo decay rate, 1/T-2(CPMG) as a function of the repetition rate, 1/t(cp), of the refocusing pulses. Expressions describing the dependence of T-2(CPMG) as a function of 1/t(cp) and k(-1) have been previously derived with sufficient generality to include the two-site inhibitor-enzyme exchange case. Existing expressions for T-1 rho as a function of k(ex) and omega(SL), however, had to be reformulated to take into account differences between T-2(b) and T-1(b) for the bound form of the inhibitor as well as offset corrections important at low values of omega(SL) A new expression for exchange-mediated T-1 rho has been derived to take these factors into account and is shown to provide a more accurate description of observed T-1 rho data than previous models. Numerical analysis of relaxation rates, measured independently by either the rotating-frame or the spin-echo method for the H-1 and H-2 protons of tubercidin at different inhibitor:enzyme ratios, yields comparable values for k(-1) of 2400 (+/-350) and 900 (+/-80) s(-1) at 20 and 10 degrees C, respectively. The merits of both methods are compared and suggestions for optimizing the experiments are discussed. (C) 1994 Academic Press, Inc.