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. 1996 Feb 20;35(7):2201-8.
doi: 10.1021/bi951883n.

Contribution of facilitated diffusion and processive catalysis to enzyme efficiency: implications for the EcoRI restriction-modification system

M A Surby  1 , N O Reich
Affiliations

Contribution of facilitated diffusion and processive catalysis to enzyme efficiency: implications for the EcoRI restriction-modification system

M A Surby et al. Biochemistry. .

Abstract

The contribution of nonspecific DNA to enzyme efficiency (k(cat)/K(m)) is described for a sequence-specific DNA-modifying enzyme. Our investigation focuses on the EcoRI DNA methyltransferase which transfers a methyl group from the cofactor S-adenosylmethionine to the second adenine in the double-stranded DNA sequence GAATTC. k(cat)/K(m) increases 4-fold 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 from 29 to 378 base pairs. No changes in k(cat)/K(m) result from further increases in either case. A facilitated diffusion mechanism is proposed in which the methyltransferase scans an average of <400 base pairs prior to dissociation from a DNA molecule. The methyltransferase was found to methylate two sites on a single DNA molecule in a distributive rather than a processive manner, suggesting that the enzyme dissociates from the DNA prior to release of the reaction product S-adenosylhomocysteine. A direct competition experiment with the EcoRI endonuclease shows the methyltransferase to be slightly more efficient at specific site location and catalysis. A rationale for the role of facilitated diffusion in this type II restriction-modification system is proposed.

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