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

FEBS Letters

Struminskaya, Nina K.²  Yakovlev, Gennady I.²  Leschinskaya, Inna B.¹  Chernokalskaya, Elena B.³  Kipenskaya, Larisa V.¹  Borzykh, Oleg A.²  Znamenskaya, Lelya V.¹  Hartley, Robert W.³  Moiseyev, Gennady P.² 

[1] Kazan State University, Biological Department, Kazan 420008, Russian Federation;Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 117984, Russian Federation;Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA

关键词: Ribonuclease;    Catalytic property;    Site-directed mutagenesis;    3′GMP;    guanosine 3′-phosphate;    GpA;    guanylyl- (3′–5′)adenosine;    poly(I);    polyinosinic acid;    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);   

DOI  :  10.1016/0014-5793(94)01150-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 His¹⁰¹Glu 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 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 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.

【 授权许可】

Unknown