We carried out a steady state kinetic analysis of the @entity1519 DNA-[N6- @entity5536 ]methyltransferase ( @entity1519 Dam) mediated @entity5498 group transfer reaction from @entity3874 ( @entity7726 ) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and @entity55851 , AdoHcy) release was @entity7726 downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate @entity7726 concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free @entity1519 Dam (initially in conformational form E) randomly interacts with substrates @entity7726 and DNA to form a ternary @entity1519 Dam- @entity7726 -DNA complex in which @entity1519 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of @entity5498 group transfer from @entity7726 to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1). After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind @entity7726 (unlike form E, which randomly binds @entity7726 and DNA), and the @entity7726 -F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of @entity7726 in a single concerted event, while @entity1519 Dam remains in the isomerized form F. The resulting @entity7726 -F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high @entity7726 concentrations.

@entity1519 Dam DNA-[N6- XXXX ]methyltransferase. Kinetic evidence for a catalytically essential conformational change in the ternary complex.

@entity55851 :: ('CHEBI:57856', 'Chemical') :: ['S-adenosyl-l-homocysteine'] @entity3874 :: ('CHEBI:15414', 'Chemical') :: ['S-adenosyl-l-methionine'] @entity5536 :: ('MESH:D000225', 'Chemical') :: ['adenine'] @entity7726 :: ('MESH:D012436', 'Chemical') :: ['AdoMet'] @entity5498 :: ('CHEBI:32875', 'Chemical') :: ['methyl'] @entity1519 :: ('10665', 'Species') :: ['bacteriophage T4', 'T4']
@entity5536 :: (MESH:D000225,Chemical) :: ['adenine']
