Comparative study of binase and barnase: experience in chimeric ribonucleases.
Chimeric enzymes were constructed to elucidate the differences in physicochemical properties of two related bacterial RNases, barnase and binase. Chimeras (Ba26Bi, Ba73Bi, Ba26Bi73Ba and Bi73Ba) contain six to thirteen residue substitutions relative to barnase, which are beyond the active site. The catalytic activity of RNases toward GpU, GpC and poly(I), as well as conformational distinctions and heat denaturation parameters, were studied. Thermal denaturation of binase, barnase and chimeric RNases is a two-state transition. The mutation-induced changes in the free energy of unfolding of barnase deduced from thermal and urea denaturation nearly coincide. The kinetic parameters for GpU and GpC demonstrate that the chimeras fall into two groups: barnase-like and binase-like. This division is determined by the origin of their C-terminal part (residues 73-110) which is also responsible for their thermostability at pH 2.4. An inverse linear dependence was found between kcat for poly(I) and denaturation temperature of RNases at pH 5.5, which points out that certain lability of the protein molecule appears to be necessary for efficient polynucleotide cleavage.
Schulga
A
A
Centre Bioengineering, Russian Academy of Sciences, Moscow.
Kurbanov
F
F
Kirpichnikov
M
M
Protasevich
I
I
Lobachov
V
V
Ranjbar
B
B
Chekhov
V
V
Polyakov
K
K
Engelborghs
Y
Y
Makarov
A
A
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D004722:Endoribonucleases; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; C022543:ribonuclease T(2)