1bir

From Proteopedia

(Difference between revisions)

Revision as of 09:55, 21 February 2008

RIBONUCLEASE T1, PHE 100 TO ALA MUTANT COMPLEXED WITH 2' GMP

Overview

The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.

About this Structure

1BIR is a Single protein structure of sequence from Aspergillus oryzae with and as ligands. Active as Ribonuclease T(1), with EC number 3.1.27.3 Full crystallographic information is available from OCA.

Reference

A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1., Doumen J, Gonciarz M, Zegers I, Loris R, Wyns L, Steyaert J, Protein Sci. 1996 Aug;5(8):1523-30. PMID:8844843

Page seeded by OCA on Thu Feb 21 11:55:48 2008

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/1bir"

@@ Line 1: / Line 1: @@
-[[Image:1bir.jpg|left|200px]]<br /><applet load="1bir" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1bir.jpg|left|200px]]<br /><applet load="1bir" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1bir, resolution 1.8&Aring;" />
 '''RIBONUCLEASE T1, PHE 100 TO ALA MUTANT COMPLEXED WITH 2' GMP'''<br />
 ==Overview==
-The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3), has been investigated by site-directed mutagenesis and X-ray, crystallography. Replacement of Phe 100 by alanine results in a mutant, enzyme with kcat reduced 75-fold and a small increase in Km for the, dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has, similar effects on the turnover rates of GpC and its minimal analogue, GpOMe, in which the leaving cytidine is replaced by methanol. The, contribution to catalysis is independent of the nature of the leaving, group, indicating that Phe 100 belongs to the primary site. The, contribution of Phe 100 to catalysis may result from a direct van der, Waals contact between its aromatic ring and the phosphate moiety of the, substrate. Phe 100 may also contribute to the positioning of the, pentacovalent phosphorus of the transition state, relative to other, catalytic residues. If compared to the corresponding wild-type data, the, structural implications of the mutation in the present crystal structure, of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are, restricted to the active site. Repositioning of 2'-GMP, caused by the Phe, 100 Ala mutation, generates new or improved contacts of the phosphate, moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58, carboxylate appear to be weakened. The effects of the His 92 Gln and Phe, 100 Ala mutations on GpC turnover are additive in the corresponding double, mutant, indicating that the contribution of Phe 100 to catalysis is, independent of the catalytic acid His 92. The present results lead to the, conclusion that apolar residues may contribute considerably to catalyze, conversions of charged molecules to charged products, involving even more, polar transition states.
+The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.
 ==About this Structure==
-BIR is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aspergillus_oryzae Aspergillus oryzae] with CA and 2GP as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Ribonuclease_T(1) Ribonuclease T(1)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.27.3 3.1.27.3] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1BIR OCA].
+BIR is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Aspergillus_oryzae Aspergillus oryzae] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=2GP:'>2GP</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Ribonuclease_T(1) Ribonuclease T(1)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.27.3 3.1.27.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1BIR OCA].
 ==Reference==
@@ Line 26: / Line 26: @@
 [[Category: nuclease]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 11:41:04 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:55:48 2008''

1bir

From Proteopedia

Revision as of 09:55, 21 February 2008

Overview

About this Structure

Reference

Proteopedia Page Contributors and Editors (what is this?)

Views

Personal tools

Navigation

Search

Toolbox