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.
Reich
N O
NO
Department of Chemistry, University of California, Santa Barbara 93106.
Mashhoon
N
N
D000225:Adenine; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012436:S-Adenosylmethionine; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000225:Adenine