Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

Abstract

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII-DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metalion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the twometal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

Mg2+ions: do they bind to nucleobase nitrogens?

“…Given the affinity of Mn 2+ for N7, this transition metal could eventually replace Mg 2+ but more probably also Na + commending great care in the interpretation of ion substitution experiments. This is not purely speculative since we are aware of at least one example of substitution of Na + by Mn 2+ in a protein crystal structure ( 118 ).…”