CONTRIBUTION OF FACILITATED DIFFUSION AND PROCESSIVE CATALYSIS TO ENZYME EFFICIENCY - IMPLICATIONS FOR THE ECORI RESTRICTION-MODIFICATION SYSTEM

The contribution of nonspecific DNA to enzyme efficiency (k(cat)/K-m) is described for a sequence-specific DNA-modifying enzyme. Our investi gation focuses on the EcoRI DNA methyltransferase which transfers a me thyl group from the cofactor S-adenosylmethionine to the second adenin e in the double-stranded DNA sequence GAATTC, k(cat)/K-m increases 4-f old as DNA length increases from 14 to 429 base pairs and increases 2- fold as the distance from the site to the nearest end is increased fro m 29 to 378 base pairs. No changes in k(cat)/K-m result from further i ncreases in either case. A facilitated diffusion mechanism is proposed in which the methyltransferase scans an average of <400 base pairs pr ior to dissociation from a DNA molecule. The methyltransferase was fou nd to methylate two sites on a single DNA molecule in a distributive r ather than a processive manner, suggesting that the enzyme dissociates from the DNA prior to release of the reaction product S-adenosylhomoc ysteine. A direct competition experiment with the EcoRI endonuclease s hows the methyltransferase to be slightly more efficient at specific s ite location and catalysis. A rationale for the role of facilitated di ffusion in this type II restriction-modification system is proposed.