Mutational analysis of the active site of RNase of Bacillus intermedius (BINASE)

• Published in: FEBS letters 1994-11, Vol.354 (3), p.305-306
• Main Authors: Yakovlev, Gennady I., Moiseyev, Gennady P., Struminskaya, Nina K., Borzykh, Oleg A., Kipenskaya, Larisa V., Znamenskaya, Lelya V., Leschinskaya, Inna B., Chernokalskaya, Elena B., Hartley, Robert W.
• Format: Article
• Language: English
• Subjects: 3′GMP, guanosine 3′-phosphate; Bacillus - enzymology; Bacillus intermedius; Binding Sites; Catalytic property; DNA Mutational Analysis; Endoribonucleases - chemistry; Endoribonucleases - genetics; Endoribonucleases - metabolism; Glutamic Acid - chemistry; GpA, guanylyl- (3′–5′)adenosine; Histidine - chemistry; Hydrogen-Ion Concentration; Kinetics; Mutagenesis, Site-Directed; poly(A), polyadenylic acid. Enzymes: ribonucleases binase from Bacillus intermedius and barnase from Bacillus amyloquefaciens (EC 3.1.4.23), ribonuclease A, bovine pancreatic ribonuclease (EC 3.1.27.5); poly(I), polyinosinic acid; Ribonuclease; Site-directed mutagenesis; Structure-Activity Relationship
• ISSN: 0014-5793; 1873-3468
• 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 His 101Glu is 2.0–2.7% of that for native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant k cat with almost unchanged affinity of the enzyme for the substrate, characterized by K M. This is the expected result if His 101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys 26 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 k cat and K M for poly(I) and poly(A) but in k cat 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.