Contribution of arginine‐82 and arginine‐86 to catalysis of RNases from Bacillus intermedius (binase)

FEBS Letters

Hartley, Robert W²  Struminskaya, Nina K¹  Kipenskaya, Larisa V³  Leschinskaya, Inna B³  Yakovlev, Gennady I¹  Znamenskaya, Lilia V³ 

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

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;   

DOI  :  10.1016/S0014-5793(98)00485-2

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 Arg⁸² and Arg⁸⁶ in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg⁸⁶Ala is 2.7×10³–7.7×10³ times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant k _cat with a relatively small change of enzyme-substrate affinity, characterized by K _m. This is the expected result if Arg⁸⁶ acts to lower the energy of a transition state of the reaction. The replacement of Arg⁸² by Ala causes a 5–19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg⁸² by alanine is mediated by the effect of Arg⁸² on the pK of catalytic residues.

【 授权许可】

Unknown