1axe: Difference between revisions

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CRYSTAL STRUCTURE OF THE ACTIVE-SITE MUTANT PHE93->TRP OF HORSE LIVER ALCOHOL DEHYDROGENASE IN COMPLEX WITH NAD AND INHIBITOR TRIFLUOROETHANOL

OverviewOverview

We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25 degrees C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 --> Trp) and a low-tunneling (Val-203 --> Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 --> Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 --> Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.

About this StructureAbout this Structure

1AXE is a Single protein structure of sequence from Equus caballus with , and as ligands. Active as Alcohol dehydrogenase, with EC number 1.1.1.1 Full crystallographic information is available from OCA.

ReferenceReference

A link between protein structure and enzyme catalyzed hydrogen tunneling., Bahnson BJ, Colby TD, Chin JK, Goldstein BM, Klinman JP, Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12797-802. PMID:9371755

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-[[Image:1axe.gif|left|200px]]<br /><applet load="1axe" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1axe.gif|left|200px]]<br /><applet load="1axe" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1axe, resolution 2.0&Aring;" />
 '''CRYSTAL STRUCTURE OF THE ACTIVE-SITE MUTANT PHE93->TRP OF HORSE LIVER ALCOHOL DEHYDROGENASE IN COMPLEX WITH NAD AND INHIBITOR TRIFLUOROETHANOL'''<br />
 ==Overview==
-We present evidence that the size of an active site side chain may, modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction., Primary and secondary kH/kT and kD/kT kinetic isotope effects have been, measured for the oxidation of benzyl alcohol catalyzed by horse liver, alcohol dehydrogenase at 25 degrees C. As reported in earlier studies, the, relationship between secondary kH/kT and kD/kT isotope effects provides a, sensitive probe for deviations from classical behavior. In the present, work, catalytic efficiency and the extent of hydrogen tunneling have been, correlated for the alcohol dehydrogenase-catalyzed hydride transfer among, a group of site-directed mutants at position 203. Val-203 interacts with, the opposite face of the cofactor NAD+ from the alcohol substrate. The, reduction in size of this residue is correlated with diminished tunneling, and a two orders of magnitude decrease in catalytic efficiency. Comparison, of the x-ray crystal structures of a ternary complex of a high-tunneling, (Phe-93 --&gt; Trp) and a low-tunneling (Val-203 --&gt; Ala) mutant provides a, structural basis for the observed effects, demonstrating an increase in, the hydrogen transfer distance for the low-tunneling mutant. The Val-203, --&gt; Ala ternary complex crystal structure also shows a hyperclosed, interdomain geometry relative to the wild-type and the Phe-93 --&gt; Trp, mutant ternary complex structures. This demonstrates a flexibility in, interdomain movement that could potentially narrow the distance between, the donor and acceptor carbons in the native enzyme and may enhance the, role of tunneling in the hydride transfer reaction.
+We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25 degrees C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 --&gt; Trp) and a low-tunneling (Val-203 --&gt; Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 --&gt; Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 --&gt; Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.
 ==About this Structure==
-AXE is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Equus_caballus Equus caballus] with ZN, NAD and ETF as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alcohol_dehydrogenase Alcohol dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.1 1.1.1.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1AXE OCA].
+AXE is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Equus_caballus Equus caballus] with <scene name='pdbligand=ZN:'>ZN</scene>, <scene name='pdbligand=NAD:'>NAD</scene> and <scene name='pdbligand=ETF:'>ETF</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alcohol_dehydrogenase Alcohol dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.1 1.1.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1AXE OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Equus caballus]]
 [[Category: Single protein]]
-[[Category: Chin, J.K.]]
+[[Category: Chin, J K.]]
-[[Category: Colby, T.D.]]
+[[Category: Colby, T D.]]
-[[Category: Goldstein, B.M.]]
+[[Category: Goldstein, B M.]]
 [[Category: ETF]]
 [[Category: NAD]]
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 [[Category: oxidoreductase (nad(a)-choh(d))]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 11:11:06 2007''
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