Investigation of ionizable residues critical for sequence-specific enzymatic DNA modification: protein modification and steady-state and pre-steady-state kinetic pH analyses of EcoRI DNA methyltransferase.
Steady-and pre-steady-state pH kinetic analyses are widely used methods to investigate important ionizable groups in enzyme-catalyzed reactions. The first such analysis to identify ionizable residues critical for sequence-specific modification of DNA is presented. EcoRI DNA methyltransferase uses S-adenosyl-L-methionine (AdoMet) to catalyze the N6 methylation of the second adenine in the double-stranded DNA sequence GAATTC. The kinetic mechanism was previously shown to be steady-state-ordered bi bi in which AdoMet binds first followed by DNA addition [Reich, N. O., & Mashhoon, N. (1991) Biochemistry 30, 2933-2939]. Steady-state parameters are strongly dependent on pH and implicate at least four residues with pKa values between 8.2 and 8.9 in the free enzyme and AdoMet-Bound enzyme and one residue with an apparent pKa of 6.0. The data obtained are consistent with the enzyme binding the form of AdoMet in which the alpha amino group is protonated. Two protein residues with an apparent pKa between 8.9 and 9.2 were implicated within the central complex (enzyme-DNA-AdoMet). The general insensitivity of all steady-state parameters to pH changes between pH 6.0 and 8.0 suggests that no critical protein residues undergo ionization-state changes in this range. The lack of significant pH-dependent changes in protein fluorescence and DNA thermal stability suggests minimal structural changes in either macromolecule. In support of the steady-state results single-turnover experiments reveal minimal pH dependence of the methylation rate constant between pH 5.53 and 8.6. Thus, no amino acids critical for catalysis undergo ionization-state changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)
Mashhoon
N
N
Department of Chemistry, University of California, Santa Barbara 93106.
Reich
N O
NO
D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011506:Proteins / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D007477:Ions; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D003545:Cysteine; D005033:Ethylmaleimide