Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis.
Endonuclease V, a pyrimidine dimer-specific DNA repair enzyme, was chemically modified by reductive methylation, a technique that specifically methylates primary amino groups. Upon reaction of endonuclease V with [14C]formaldehyde (14CH2O) in the presence of the reducing agent sodium cyanoborohydride (Na-CNBH3), it was discovered that 0.8 methylation/endonuclease V molecule was required to reduce both the glycosylase and the phosphodiester lyase activities by 70-80%. Pyrimidine dimer-specific binding was not eradicated at a level of methylation equivalent to 0.8 CH3/endonuclease V molecule but was eradicated at higher levels of methylation. Endonuclease V that had been modified with an average of 1.6 CH3/molecule was digested with Staphylococcus aureus strain V8 protease and the peptides subsequently separated by reverse-phase high performance liquid chromatography. Radiolabel was found exclusively on the peptide including the amino terminus, as determined by the percent amino acid composition. Neither intact CH3-endonuclease V nor radiolabeled peptides were able to be sequenced by Edman degradation indicating blockage of the amino terminus by methylation. This study shows strong evidence for the unusual involvement of the alpha NH2 moiety in the chemical mechanisms of endonuclease V. A reaction mechanism that incorporates these findings is presented.
Schrock
R D
RD
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Lloyd
R S
RS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data
D000596:Amino Acids; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer)