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PDBsum entry 1m0h
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References listed in PDB file
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Key reference
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Title
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Residues involved in the catalysis, Base specificity, And cytotoxicity of ribonuclease from rana catesbeiana based upon mutagenesis and x-Ray crystallography.
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Authors
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Y.J.Leu,
S.S.Chern,
S.C.Wang,
Y.Y.Hsiao,
I.Amiraslanov,
Y.C.Liaw,
Y.D.Liao.
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Ref.
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J Biol Chem, 2003,
278,
7300-7309.
[DOI no: ]
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PubMed id
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Abstract
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The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A
superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active
among frog ribonucleases, has a unique base preference for pyrimidine-guanine
rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base
specificity and catalytic activity were determined by site-directed mutagenesis,
k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA
substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and
Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic
activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base
specificity. The cytotoxicity of RC-RNase is correlated, but not proportional
to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA)
complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and
His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9
was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38.
Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines
B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form
four hydrogen bonds with guanine at B(2) site and determine B(2) base
specificity.
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Figure 5.
Fig. 5. Ribbon diagram of the three-dimensional structure
of two RC-RNase·d(ACGA) complex molecules. Two d(ACGA)
molecules are shown by liquorice representation viewing down
along the noncrystallographic 2-fold axis. The  -helix and
 -sheet
strand are colored green and blue, respectively. The base
stacking and continuity of the phosphate backbone are clearly
seen in the central area of the diagram.
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Figure 7.
Fig. 7. Stereo diagrams of RC-RNase and oligonucleotide
complex. A, ribbon diagram of RC-RNase with d(CG), the -helix,
-sheet, and
d(CG) are shown in green, blue, and red, respectively. The
relevant residues in the B[1], B[2], and P[1] sites are
numbered. B, C, and D show the hydrogen bonds between respective
residues and substrates in B[1], B[2], and P[1] sites,
respectively. The hydrogen bonds are shown as blue dashed lines.
The substrate in red was shown within the electronic density
omit map (contoured at 1.5  ). The
image was generated by SwissPDBviewer (50), Turbo-Frodo, and
Photoshop (Adobe Systems, Inc.).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2003,
278,
7300-7309)
copyright 2003.
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