Direct measurements of the dissociation-rate constant for inhibitor-enzyme complexes via the T1 rho and T2 (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 1H NMR methods for studying exchange-mediated transverse relaxation. One method involves measurements of the decay rate, 1/T1 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/TCPMG2, as a function of the repetition rate, 1/tcp, of the refocusing pulses. Expressions describing the dependence of TCPMG2 as a function of 1/tcp and k-1 have been previously derived with sufficient generality to include the two-site inhibitor-enzyme exchange case. Existing expressions for T1 rho as a function of kex and omega SL, however, had to be reformulated to take into account differences between Tb2 and Tb1 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 T1 rho has been derived to take these factors into account and is shown to provide a more accurate description of observed T1 rho data than previous models. Numerical analysis of relaxation rates, measured independently by either the rotating-frame or the spin-echo method for the H1' and H2 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Davis
D G
DG
Laboratory of Molecular Biophysics, NIEHS, Research Triangle Park, North Carolina 27709.
Perlman
M E
ME
London
R E
RE
D009682:Magnetic Resonance Spectroscopy; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D014372:Tubercidin / Q000737:chemistry*
D011683:Purine-Nucleoside Phosphorylase; D014372:Tubercidin