Crystallographic Studies on Structural Features That Determine the Enzymatic Specificity and Potency of Human Angiogenin: Thr44, Thr80, and Residues 38-41
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- 作者：Daniel E. Holloway ; Gayatri B. Chavali ; Michelle C. Hares ; Matthew D. Baker ; Gowtham V. Subbarao ; Robert Shapiro ; and K. Ravi Acharya
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：February 10, 2004
- 年：2004
- 卷：43
- 期：5
- 页码：1230 - 1241
- 全文大小：691K
- 年卷期：v.43,no.5(February 10, 2004)
- ISSN：1520-4995
文摘
Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member ofthe pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavageafter cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristicactivity, we have determined the crystal structures of three Ang variants having novel activity. (i) Thestructure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that theintrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of thisvariant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen inany Ang variant thus far. This allows the side chain of Arg101 to penetrate the B1 site, raising the possibilitythat it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supportsthe view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus andthat its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang arereplaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segmentinfluences catalysis by subtle means, possibly by reducing the pKa of the catalytic lysine. The loss ofangiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sitesbut may be a consequence of the chimera's enhanced ribonucleolytic activity.