MECHANISTIC STUDIES OF ANTIBODY-CATALYZED PYRIMIDINE DIMER PHOTOCLEAVAGE

An antibody elicited against the trans, syn uracil cyclobutane dimer h apten 1 catalyzes the light-dependent cleavage of uracil dimers 1 and 2 to the corresponding monomers 3 and 4. Kinetic analysis of the antib ody-catalyzed reaction affords a value of k(cat)/K-M = 1.7 x 10(3) M(- 1) min(-1) for substrate 2, and comparison to the uncatalyzed reaction gives a rate acceleration of k(cat)/k(uncat) = 380. The wavelength de pendence of the reaction and fluorescence quenching behavior suggest t hat a tryptophan residue is acting as a photosensitizer. The reaction mechanism was probed by measurement of secondary deuterium isotope eff ects. Substrates with selective deuterium substitutions in the cyclobu tane ring were prepared, and isotope effects were measured by the meth od of internal competition using electrospray-ionization mass spectrom etry to quantify the products. Kinetic isotope effects of (alpha-D)(V/ K) = 1.11, 1.14, and 1.20 were observed for the 5,5'-, 6,6'-, and 5,5' ,6,6'-labeled substrates, respectively. These results are comparable t o those observed in a similar study on the E. coli enzyme DNA photolya se(1) and suggest that the reaction may proceed via a radical anion in termediate with concerted breakage of the 5,5' and 6,6' bonds, Alterna tively the reaction may proceed via a mechanism in which the first bon d is cleaved in a reversible fashion.