Effects of non-conservative changes to tyrosine 76, a key DNA binding residue of DNase I, on phosphodiester bond cleavage and DNA hydrolysis selectivity.
Non-conservative changes, consisting of Y76E, Y76L, Y76Q and Y76W, have been made to tyrosine 76, one of the key DNA binding residues in DNase I. Normally Y76 inserts into the minor groove of DNA and makes an unusual, hydrophobic, stacking interaction with one of the sugars. All four mutants bind to DNA more tightly than the wild type, but cut it more slowly as assessed by Kunitz assays. This gives a rather small decrease in the specificity constants (Vmax/K(m)) for the hydrolysis of DNA, which is roughly paralleled by the loss of activity towards the non-DNA small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)phosphate. These non-conservative mutants, therefore, show different behaviour to Y76A and Y76G, studied previously [Doherty A.J., Worrall A.F. and Connolly B.A. (1995) J: Mol. Biol., 251, 366-377]. These two mutants both bind to and cut DNA poorly, resulting in large decreases in Vmax/K(m) values. However, they showed little reduction in rates with the chromophoric substrate. It is likely that the altered side chains in the non-conservative mutants are still able to interact productively with the DNA and contribute to the observed DNA distortion that is essential for efficient catalysis. However, these mutations disrupt the active site, most probably by interference with the hydrogen bonded Y76-E78-H134 triad. H134 is a critical hydrolytic residue of DNase I that is essential for catalysis. The DNA cleavage selectivity of the Y76E, Y76L, Y76Q and Y76W mutants were little altered as compared with the wild-type enzyme as measured using the cutting patterns of a 160 base-pair Escherichia coli Tyr T promoter DNA fragment. This confirms earlier observations, with Y76F, Y76A and Y76G, that showed that this tyrosine has little role in DNA cleavage specificity.
Warren
M A
MA
Department of Biochemistry and Genetics, University of Newcastle, Newcastle upon Tyne, UK.
Evans
S J
SJ
Connolly
B A
BA
D001345:Autoradiography; D004247:DNA / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D004247:DNA; D003850:Deoxyribonuclease I