Yakovlev G I, Moiseyev G P, Struminskaya N K, Borzykh O A, Kipenskaya L V, Znamenskaya L V, Leschinskaya I B, Chernokalskaya E B, Hartley R W
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
FEBS Lett. 1994 Nov 14;354(3):305-6. doi: 10.1016/0014-5793(94)01150-8.
To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101 Glu is 2.0-2.7% of that for the native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant kcat, with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both kcat and KM for poly(A) and poly(A) but in kcat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.
