Role of the reactive cysteine residue in restriction endonuclease Cfr9I.
Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.
Siksnys
V
V
Institute of Biotechnology Fermentas, Vilnius, Lithuania.
Pleckaityte
M
M
D001483:Base Sequence; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D004262:DNA Restriction Enzymes / Q000037:antagonists & inhibitors / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008969:Molecular Sequence Data; D009841:Oligonucleotides
D007461:Iodoacetates; D009841:Oligonucleotides; D004228:Dithionitrobenzoic Acid; D004262:DNA Restriction Enzymes; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D003545:Cysteine; D005033:Ethylmaleimide; D019807:Iodoacetic Acid