Aspartic acid‐121 functions at the active site of bovine pancreatic ribonuclease

Stern, Mary S.; Doscher, Marilynn S.

doi:10.1016/0014-5793(84)80498-6

Cited by 24 publications

(14 citation statements)

References 21 publications

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“…Our kinetic results can be compared with the results obtained with the 'semi-synthetic' proteins formed by non-covalent complexation between RNase l-120 and 6"' f-,:ious synthetic peptides [15,16]. With Asn-121 in the peptide, the turnover catalyzed by the complex is 4.5% of the catalytic activity of the com- (Table I), Indeed, although the measured rates are quite low and therefore imprecise, it appears that the double mutant has a higher activity than the K4 IR mutant itself under at Ieast some assa~ conditions (e,g.…”

Section: Resultsmentioning

confidence: 99%

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Site‐directed mutagenesis of bovine pancreatic ribonuclease: Lysine‐41 and aspartate‐121

et al. 1991

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Chemical modification studies suggest that two residues of bovine pancreatic ribonuclease A (RNase A), Lys‐41 and Asp‐121, are important for catalysis. Three mutants of RNase A have been prepared, two point mutants with Lys‐41 altered to Arg‐41 and Asp‐121 altered to Glu‐121, and a double mutant where both residues are altered. The Lys‐41 Arg mutant has ca. 2% the calatylic activity (kcalKm ) of the native protein, while the Asp‐121 Glu mutant has ca. 17% the catalytic activity of the native protein. The double mutant has catalytic activity comparable to the Lys‐41 Arg mutant.

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Section: Resultsmentioning

confidence: 99%

“…When the C-terminal residues of RNase (residues 121-124) are removed by proteolysis, only 0.5% to 4070 of the catalytic activity towards various substrates is retained [14,lSl; further digestion removes all traces of catalytic activity. However, catalytic activity can be restored by adding synthetic peptides extending from residues Ill-124 [16] [ 19). …”

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confidence: 99%

Site‐directed mutagenesis of bovine pancreatic ribonuclease: Lysine‐41 and aspartate‐121

et al. 1991

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Section: Discussionmentioning

confidence: 99%

“…Semisynthetic RNases (18,19) were subsequently used to examine the functional roles of various residues near the C terminus, including Asp-121, which is fully conserved (20)(21)(22). Its replacement by asparagine decreases catalysis by a factor of 20 but does not significantly change substrate binding for cytidine 2',3'-cyclic monophosphate (22). A hydrogen bond between the p-carboxylate of Asp-121 and the imidazole of His-119 in crystal structures of both RNase bound to a substrate analogue (11) and the free enzyme (12,13) bond, placing the Nff of His-119 within hydrogen bonding distance of the substrate phosphate group (23).…”

Section: Discussionmentioning

confidence: 99%

Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin.

Harper

Vallée²

1988

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT, Site-specific mutagenesis of the blood vesselinducing protein angiogenin has been used to further explore both its homology to pancreatic ribonuclease and the functional roles of particular residues. Replacement of Asp-116 in angiogenin by either asparagine (D116N), alanine (D116A)f or histidine (D116H) markedly enhances both its ribonucleolytic activity and angiogenic potency. Activity toward tRNA is -8-, 15-, and 18-fold greater than native angiogenin for D116N-, D116A-, and.D116H-angiogenin, respectively. The enzymatic specificity of angiogenin, however, has been maintained. Thus, cleavage of 18S and 28S rRNA by the most active His-116 mutant yields-the same pattern of polynucleotide products as from angiogenin, whereas there are only minor alterations in activity with cytidylyl(3',5')adenosine and uridylyl(3',5')-adenosine. Extensive biological assays on the chicken embryo chorioallantoic membrane demonstrate that D116H-angiogenin is one to two orders ofmagnitude more potent in inducing neovascularization than native angiogenin, which correlates well with enhanced enzymatic action. These results support the proposition that the enzymatic and angiogenic activities on angiogenin are interrelated.

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“…(19). On the basis of structural studies, these residues as well as glutamine-1l and aspartate-121 of RNase have been proposed as being important in the binding of substrate (13,20). All of these residues are conserved in the angiogenin molecule.…”

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confidence: 99%

A preliminary three-dimensional structure of angiogenin.

Palmer¹,

Scheraga²,

Riordan³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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A preliminary three-dimensional structure of angiogenin has been computed, based on its homology to bovine pancreatic ribonuclease A. A standard-geometry structure of ribonuclease was first obtained from its x-ray coordinates. The fit of the backbone of angiogenin to that of ribonuclease was then optimized by taking account of amino acid deletions and by minimizing its conformational energy-plus-a-penalty distance function constraining its backbone to that of ribonuclease. Side-chain and backbone dihedral angles were allowed to vary throughout the cycles of energy minimization. In the last stages of minimization, the penalty distance function was removed. A low-energy structure resembling ribonuclease was obtained.Angiogenin is a basic single-chain protein of 123 amino acid residues that induces in vivo angiogenesis, the formation of blood vessels and a vascular system (1-3). The protein has been isolated in pure form from the serum-free conditioned medium of the human colorectal adenocarcinoma cell line HT-29. Both the amino acid sequence (2) and the corresponding DNA sequence (3) have been determined, showing it to have about a 35% homology with several mammalian pancreatic ribonucleases. Since the structure of bovine pancreatic ribonuclease is known (4, 5), the structure of angiogenin can be computed by minimization of its conformational energy on the assumption that it has a three-dimensional backbone structure similar to that of ribonuclease. This approach is used here to compute a preliminary three-dimensional structure of the whole angiogenin molecule. A similar procedure has been used to compute the structure of bovine a-lactalbumin (6) from that of hen egg-white lysozyme (7) and those of several snake venom inhibitors (8) from that of bovine pancreatic trypsin inhibitor (9). METHODOLOGY AND RESULTSOptimized Structure of Ribonuclease. First, a model of ribonuclease (RNase) having the standard geometry (bond lengths and bond angles) of the ECEPP (empirical conformational energy program for peptides) algorithm (10, 11) was computed from the x-ray coordinates (4, 5). The objective was to obtain an energy-minimized, standard-geometry structure for ribonuclease that would superimpose well on the known 2.0-A resolution x-ray structure and be free of any high-energy atomic overlaps. The coordinates from this standard-geometry model could then be used as input to provide a starting conformation for the backbone of angiogenin, as well as for side chains that are conserved between the two molecules. The degrees of freedom in this type of model are dihedral angles, with bond lengths and bond angles fixed at values obtained from high-resolution crystal structures of small molecules. For those dihedral angles that could be defined by the positions of four heavy atoms, initial values were computed from the neutron-and x-ray-derived Cartesian coordinates (4, 5). For the initial ECEPP model, all peptide bond dihedral angles, cl, were fixed at 1800 with the exception of those preceding proline residues, which wer...

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Aspartic acid‐121 functions at the active site of bovine pancreatic ribonuclease

Cited by 24 publications

References 21 publications

Site‐directed mutagenesis of bovine pancreatic ribonuclease: Lysine‐41 and aspartate‐121

Site‐directed mutagenesis of bovine pancreatic ribonuclease: Lysine‐41 and aspartate‐121

Mutagenesis of aspartic acid-116 enhances the ribonucleolytic activity and angiogenic potency of angiogenin.

A preliminary three-dimensional structure of angiogenin.

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