KINETIC ISOTOPE EFFECT CHARACTERIZATION OF THE TRANSITION-STATE FOR OXIDIZED NICOTINAMIDE ADENINE-DINUCLEOTIDE HYDROLYSIS BY PERTUSSIS TOXIN

Pertussis toxin from Bordatella pertussis catalyzes the ADP ribosylati on of several G-proteins, using NAD(+) as a substrate. In the absence of an acceptor protein, the toxin acts as a NAD(+) glycohydrolase. Per tussis toxin is one of the virulent factors for whooping cough and the refore a target for site-specific inhibitors based on the transition s tate structure. A family of kinetic isotope effects was determined for the hydrolysis reaction, using NAD(+) labeled with H-3, C-14, and N-1 5 as substrates. Primary isotope effects were 1.021 +/- 0.001 for [1'N -C-14]NAD(+) and 1.021 +/- 0.004 for [1(N)-N-15]NAD(+), and the double -primary effect of [1'N-C-14,1(N)-N-15]NAD(+) was 1.049 +/- 0.004. Sec ondary kinetic isotope effects were 1.207 +/- 0.010 for the [1'(N)-H-3 ]-, 1.144 +/- 0.005 for the [2'(N)-H-3]-, 0.989 +/- 0.001 for the [4'( N)-H-3]-, and 1.019 +/- 0.004 for the [5'(N)-H-3]NAD(+), respectively. Commitment to catalysis was excluded by isotope trapping experiments, and the experimental kinetic isotope effects were independent of pH. The measured isotope effects are therefore intrinsic. The isotope effe cts are remarkable because they indicate an oxocarbenium-like ribose r ing at the transition state but a stiffer than expected vibrational en vironment for C1' at the reaction center. On the basis of these isotop e effects, a bond order vibrational analysis was performed to locate a transition state structure consistent with the isotope effects. The k inetic isotope effects predict a residual bond order to the nicotinami de leaving group of 0.11, corresponding to a distance of 2.14 Angstrom . Participation of the water nucleophile is weak, consistent either wi th an S(N)1-like transition state with no water interaction or with th e water oxygen no closer than 3.5 Angstrom from the reaction center. T he positive charge of the ribose oxocarbenium is stabilized by delocal ization between the C1'-O4' and C1'-C2' bonds. The enzyme contacts res trict the vibrational environment of the reaction coordinate requiring increased bonding force constants for the enzyme-stabilized transitio n state. NAD(+) analogues with the nicotinamide ribose replaced by an iminoribitol ring, mimicking the flattened ribose ring of the transiti on state, are expected to be transition state inhibitors.