3ccx: Difference between revisions

Revision as of 20:08, 21 February 2008

ALTERING SUBSTRATE SPECIFICITY AT THE HEME EDGE OF CYTOCHROME C PEROXIDASE

OverviewOverview

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.

About this StructureAbout this Structure

3CCX is a Single protein structure of sequence from Saccharomyces cerevisiae with as ligand. Active as Cytochrome-c peroxidase, with EC number 1.11.1.5 Full crystallographic information is available from OCA.

ReferenceReference

Altering substrate specificity at the heme edge of cytochrome c peroxidase., Wilcox SK, Jensen GM, Fitzgerald MM, McRee DE, Goodin DB, Biochemistry. 1996 Apr 16;35(15):4858-66. PMID:8664277

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-[[Image:3ccx.jpg|left|200px]]<br /><applet load="3ccx" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:3ccx.jpg|left|200px]]<br /><applet load="3ccx" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="3ccx, resolution 2.3&Aring;" />
 '''ALTERING SUBSTRATE SPECIFICITY AT THE HEME EDGE OF CYTOCHROME C PEROXIDASE'''<br />
 ==Overview==
-Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit, unique specificities toward oxidation of small substrates. Ala-147 in CCP, is located near the delta-meso edge of the heme and along the solvent, access channel through which H2O2 is thought to approach the active site., This residue was replaced with Met and Tyr to investigate the hypothesis, that small molecule substrates are oxidized at the exposed delta-meso edge, of the heme. X-ray crystallographic analyses confirm that the side chains, of A147M and A147Y are positioned over the delta-meso heme position and, might therefore modify small molecule access to the oxidized heme, cofactor. Steady-state kinetic measurements show that cytochrome c, oxidation is enhanced 3-fold for A147Y relative to wild type, while small, molecule oxidation is altered to varying degrees depending on the, substrate and mutant. For example, oxidation of phenols by A147Y is, reduced to less than 20% relative to the wild-type enzyme, while Vmax/e, for oxidation of other small molecules is less affected by either, mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a, specific interaction for aniline has been introduced by the mutation., Stopped-flow kinetic data show that the restricted heme access in A147Y or, A147M slows the reaction between the enzyme and H202, but not to an extent, that it becomes rate limiting for the oxidation of the substrates, examined. The rate constant for compound ES formation with A147Y is 2.5, times slower than wild-type CCP. These observations strongly support the, suggestion that small molecule oxidations occur at sites on the enzyme, distinct from those utilized by cytochrome c and that the specificity of, small molecule oxidation can be significantly modulated by manipulating, access to the heme edge. The results help to define the role of, alternative electron transfer pathways in cytochrome c peroxidase and may, have useful applications in improving the specificity of peroxidase with, engineered function.
+Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.
 ==About this Structure==
-CCX is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with HEM as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cytochrome-c_peroxidase Cytochrome-c peroxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.11.1.5 1.11.1.5] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=3CCX OCA].
+CCX is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with <scene name='pdbligand=HEM:'>HEM</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cytochrome-c_peroxidase Cytochrome-c peroxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.11.1.5 1.11.1.5] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3CCX OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Saccharomyces cerevisiae]]
 [[Category: Single protein]]
-[[Category: Fitzgerald, M.M.]]
+[[Category: Fitzgerald, M M.]]
-[[Category: Goodin, D.B.]]
+[[Category: Goodin, D B.]]
-[[Category: Jensen, G.M.]]
+[[Category: Jensen, G M.]]
-[[Category: Mcree, D.E.]]
+[[Category: Mcree, D E.]]
-[[Category: Wilcox, S.K.]]
+[[Category: Wilcox, S K.]]
 [[Category: HEM]]
 [[Category: oxidoreductase (h2o2(a))]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 19:33:12 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 19:08:45 2008''