1ix8: Difference between revisions

Revision as of 14:16, 21 February 2008

Aspartate Aminotransferase Active Site Mutant V39F/N194A

OverviewOverview

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.

About this StructureAbout this Structure

1IX8 is a Single protein structure of sequence from Escherichia coli with as ligand. Active as Aspartate transaminase, with EC number 2.6.1.1 Full crystallographic information is available from OCA.

ReferenceReference

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis., Hayashi H, Mizuguchi H, Miyahara I, Nakajima Y, Hirotsu K, Kagamiyama H, J Biol Chem. 2003 Mar 14;278(11):9481-8. Epub 2002 Dec 17. PMID:12488449

Page seeded by OCA on Thu Feb 21 13:16:40 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1ix8.gif|left|200px]]<br /><applet load="1ix8" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1ix8.gif|left|200px]]<br /><applet load="1ix8" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1ix8, resolution 2.2&Aring;" />
 '''Aspartate Aminotransferase Active Site Mutant V39F/N194A'''<br />
 ==Overview==
-Aspartate aminotransferase has been known to undergo a significant, conformational change, in which the small domain approaches the large, domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a, two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258), aldimine, which has been proposed to enhance catalysis. To elucidate how, the conformational change is coupled to the shift in the aldimine pK(a), and how these changes are involved in catalysis, we analyzed structurally, and kinetically an enzyme in which Val(39) located at both the domain, interface and the entrance of the active site was replaced with a bulkier, residue, Phe. The V39F mutant enzyme showed a more open conformation, and, the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type, enzyme. When Asn(194) had been replaced by Ala in advance, the V39F, mutation did not decrease the aldimine pK(a), showing that the domain, rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal, 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the, aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type, enzyme. However, the positions of maleate, Asn(194), and Arg(386) were, superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value., The maleate-bound V39F enzyme showed an altered side-chain packing pattern, in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O, and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value., Kinetic analysis suggested that the repulsion increases the free energy, level of the Michaelis complex and promotes the catalytic reaction.
+Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.
 ==About this Structure==
-IX8 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with PLP as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Aspartate_transaminase Aspartate transaminase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.6.1.1 2.6.1.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1IX8 OCA].
+IX8 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=PLP:'>PLP</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Aspartate_transaminase Aspartate transaminase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.6.1.1 2.6.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1IX8 OCA].
 ==Reference==
@@ Line 23: / Line 23: @@
 [[Category: active site mutant]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 17:44:43 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:16:40 2008''