Contribution of Facilitated Diffusion and Processive Catalysis to Enzyme Efficiency:  Implications for the EcoRI Restriction−Modification System

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.

Simultaneous DNA Binding, Bending, and Base Flipping

“…To determine whether the FRET changes are tracking a first-or second-order process, the effect of enzyme concentration on FRET was measured. The binding kinetic constant is nearly identical to that determined independently by indirect competition methods with similar DNA sequences (16,17). Here we show that the FRET-based detection of DNA bending follows a similar pattern, in which the rate is dependent on protein concentration, but the transition clearly follows a single, well-behaved exponential curve (Fig.…”

“…3A). In the absence of either AdoMet or sinefungin, M.EcoRI binds DNA non-specifically at micromolar concentrations (16,17); under these conditions no changes in energy transfer are observed (Fig. 3A).…”

“…The enzyme uses a facilitated diffusion mechanism to locate its sites within the bacterial genome, and its specificity constant (k cat /K m ) is near diffusion-limited (16,17). The discrimination of the enzyme against single base pairmodified non-cognate site methylation is up to 20,000-fold (18).…”

“…Although no protein-DNA cocrystal structure exists for M.EcoRI, the available detailed kinetic analysis (6,(15)(16)(17)(18)(19)(20)(21) and the bending-deficient H235N mutant provide motivation for using this system to understand the relationship between DNA bending, base flipping, and catalytic specificity. Here we describe our initial efforts to determine the wild-type enzyme kinetics of bending and unbending with the cognate sequence.…”

Characterization of the LlaCI methyltransferase from Lactococcus lactis subsp. cremoris W15 provides new insights into the biology of type II restriction–modification systems

“…Clearly, it promotes DNA restriction over DNA methylation. This is facilitated by the fact that type II DNA MTases, unlike solitary enzymes such as M.EcoKDam, modify DNA in a non-processive manner (Surby & Reich, 1996;Gowher & Jeltsch, 2000;Urig et al, 2002). Our results also explain experiments that have demonstrated the recombinogenic role of restriction ENase EcoRI in vivo (Chang & Cohen, 1977;Stahl et al, 1983;Silberstein et al, 1993).…”