Presteady state kinetics of an S-adenosylmethionine-dependent enzyme. Evidence for a unique binding orientation requirement for EcoRI DNA methyltransferase.

We present the first presteady state kinetic analysis of an S-adenosylmethionine-dependent enzyme. The target enzyme is the bacterial EcoRI DNA methyl-transferase, which transfers the methyl group to the second adenine in the DNA sequence GAATTC. The rate constant for conversion of the central complex (enzyme-DNA-S-adenosylmethionine) to products (enzyme-methylated DNA-S-adenosylhomocysteine) (41 +/- 7 s-1) is over 300-fold faster than kcat, consistent with our demonstration that steps after methyl transfer are rate-limiting (Reich, N. O., and Mashhoon, N. (1991) Biochemistry 30, 2933-2939). Methyl transfer at the N6 amino moiety of adenine on each strand requires a single binding orientation.