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Enzyme
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Query: EC:3.1.27.5 (
RNase
)
17,967
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (
RNase A
). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding
RNase A
sequence, and the activities of the resulting covalent angiogenin/
RNase
hybrid, designated
ARH
-III, have been examined. The ribonucleolytic activity of
ARH
-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of
ARH
-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of
ARH
-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of
ARH
-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.
...
PMID:Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis. 169 38
The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of
pancreatic ribonuclease
(
RNase
) and contains counterparts for the critical
RNase
active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of
pancreatic RNase
by several orders of magnitude. Comparison of the amino acid sequences of
RNase
and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/
RNase
hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine
pancreatic RNase
(Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin,
ARH
-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of
ARH
-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of
pancreatic RNase
. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/
RNase
hybrid protein (ARH-IV), in which residues 59-71 of
ARH
-II have been replaced by the corresponding segment of
pancreatic RNase
, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin. 169 54
Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine
pancreatic ribonuclease
A (
RNase A
). Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than
RNase A
toward conventional substrates. A striking structural difference between angiogenin and
RNase
is the virtual absence of sequence similarity within the region of
RNase
that contains the Cys-65--Cys-72 disulfide bond. Indeed, angiogenin lacks this disulfide linkage. The present report describes the use of regional mutagenesis to generate a covalent angiogenin/
RNase
hybrid protein,
ARH
-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of
RNase A
(residues 59-73). The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds. The in vivo angiogenic potency of
ARH
-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay. In contrast, its enzymatic activity is dramatically increased. With high molecular weight wheat germ RNA and tRNA,
ARH
-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold. In addition, the specificity of
ARH
-I toward dinucleoside 3',5'-phosphates is qualitatively similar to
RNase A
; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both
RNase
and the hybrid prefer UpA to CpG.
ARH
-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis. The enhanced enzymatic properties of
ARH
-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification. The data indicate that introduction of a region of
RNase A
containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases
RNase
-like enzymatic activity while reducing its angiogenicity.
...
PMID:A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis. 271 39
Human angiogenin (Ang), a homologue of bovine
pancreatic ribonuclease
A (
RNase A
), is a potent inducer of blood vessel formation. It exerts a ribonucleolytic activity that is 10(5)-10(6)-fold lower than that of
RNase A
but nonetheless essential for biological action. Previous studies revealed some of the structural features of Ang that underlie its catalytic inefficiency: Gln-117 blocks the space corresponding to the pyrimidine binding site of
RNase A
and Ang lacks the disulfide loop 65-72 that forms most of the purine binding site of
RNase A
. Additional features have now been identified by mutagenesis and kinetics. Thr-80, which hydrogen-bonds to the pyrimidine-binding residue Thr-44, plays an important part in attenuating activity and in determining pyrimidine specificity: mutation to Ala increases activity toward cytidylyl substrates by 11-15-fold but has only a minimal effect on cleavage of uridylyl substrates. The properties of T44A/T80A and Q117A/T80A double mutants demonstrate that these changes are mediated by Thr-44 and are largely independent of the blockage by Gln-117. The side chain of Ser-118 also suppresses enzymatic activity: S118A is 5-7-fold more effective than Ang. This increase appears to reflect the loss of a hydrogen bond with Asp-116 that helps to orient Gln-117. The effects of deleting residues 119-123 suggest that main-chain atoms of the C-terminal 3(10) helix make a small further contribution. Finally, the significance of the absence of the
RNase A
loop 65-72 from Ang has been investigated by reexamining the earlier derivative
ARH
-I (in which Ang residues 58-70 have been replaced by residues 59-73 of
RNase
) and generating new derivatives of this hybrid protein. The results suggest that the
RNase A
segment of
ARH
-I not only provides more effective purine recognition but also counteracts the deleterious effects of Gln-117 and Thr-80 on the pyrimidine site.
...
PMID:Structural features that determine the enzymatic potency and specificity of human angiogenin: threonine-80 and residues 58-70 and 116-123. 957 71
Angiogenin and ribonuclease A share 33% sequence identity but have distinct functions. Angiogenin is a potent inducer of angiogenesis that is only weakly ribonucleolytic, whereas ribonuclease A is a robust
ribonuclease
that is not angiogenic. A chimera ("ARH-I"), in which angiogenin residues 58-70 are replaced with residues 59-73 of ribonuclease A, has intermediate ribonucleolytic potency and no angiogenic activity. Here we report a crystal structure of
ARH
-I that reveals the molecular basis for these characteristics. The ribonuclease A-derived (guest) segment adopts a structure largely similar to that in ribonuclease A, and successfully converts this region from a cell-binding site to a purine-binding site. At the same time, its presence causes complex changes in the angiogenin-derived (host) portion that account for much of the increased
ribonuclease
activity of
ARH
-I. Guest-host interactions of this type probably occur more generally in protein chimeras, emphasizing the importance of direct structural information for understanding the functional behavior of such molecules.
...
PMID:Guest-host crosstalk in an angiogenin-RNase A chimeric protein. 1217 35
Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the
pancreatic ribonuclease
superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii)
ARH
-II is an angiogenin/
RNase A
chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of
RNase A
. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.
...
PMID:Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41. 1475 59
