Book of Abstracts: Albany 2003
June 17-21 2003
Analysis of Contributions of the Enzyme-substrate Interactions to Purine Specificity of Barnase
Ribonuclease from Bacillus amyloliquefaciens (barnase) cleavage polynucleotides with the order of preference poly(I)>poly(A)>>poly(U)>poly(C). Crystallographic study of the barnase-3' GMP complex has shown that guanosine binding depends on hydrogen bonds between the guanine base and amino acid residues Ser57, Asn58, Arg59 and Glu60. To elucidate the contributions of induvidual hydrogen bonds between the protein atoms and the purine base of substrate to its binding, a barnase mutants ware obtained with replasements Ser57/Glu, Glu60/Ala and Glu60, Lys62/Lys, Glu and steady-state kinetics for poly(I), poly(A) and GpC cleavage were studied. The replacement of Ser57 by Glu causes a 35-fold increase in guanylic specifity in the cleavage reaction of polynucleotides. The hydrolysis rates of poly(I) and poly(A) for Glu60/Ala mutant decrease 140 and 8-fold, respectively. The catalytic activity of Glu60, Lys62/Lys, Glu barnase was the same as that of the wild type enzyme. These data support the conclusion that the hydrogen bonds between the purine base and the protein chain atoms of Asn58 and Arg59 in the enzyme-substrate complex of barnase have not essential significance. The purine preference of barnase in the hydrolysis of polynucleotide substrates is determined mainly by the hydrogen bonds between the purine base of substrate and the protein residues Ser57 and Glu60.
Engelhardt Institute of Molecular Biology