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 phosphory lase (PNPase) has been determined directly via two related H-1 NMR met hods for studying exchange-mediated transverse relaxation. One method involves measurements of the decay rate, 1/T-1 rho, of spin-locked mag netization 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(CP MG) as a function of the repetition rate, 1/t(cp), of the refocusing p ulses. Expressions describing the dependence of T-2(CPMG) as a functio n of 1/t(cp) and k(-1) have been previously derived with sufficient ge nerality to include the two-site inhibitor-enzyme exchange case. Exist ing expressions for T-1 rho as a function of k(ex) and omega(SL), howe ver, 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 observ ed T-1 rho data than previous models. Numerical analysis of relaxation rates, measured independently by either the rotating-frame or the spi n-echo method for the H-1 and H-2 protons of tubercidin at different i nhibitor: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.