1cl2: Difference between revisions

Revision as of 13:07, 21 February 2008

CYSTATHIONINE BETA-LYASE (CBL) FROM ESCHERICHIA COLI IN COMPLEX WITH AMINOETHOXYVINYLGLYCINE

OverviewOverview

The pyridoxal 5'-phosphate (PLP)-dependent cystathionine beta-lyase (CBL) was previously found to be inhibited by the natural toxins rhizobitoxine and l-aminoethoxyvinylglycine (AVG). The present study characterizes the interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) by a combination of kinetic methods and X-ray crystallography. Upon AVG treatment, time-dependent, slow-binding inhibition [Morrison, J. F. (1982) Trends Biochem. Sci. 7, 102-105] was observed due to the generation of a long-lived, slowly dissociating enzyme-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (CBL + AVG --> CBLAVG, Ki = 1.1 +/- 0.3 microM) with an association rate constant (k1) of 336 +/- 40 M-1 s-1. This value is several orders of magnitude lower than typical bimolecular rate constants of ES formation, suggesting that additional steps occur before formation of the first detectable CBLAVG complex. Loss of activity is paralleled by the conversion of the pyridoxaldimine 426 nm chromophore to a 341 nm-absorbing species. On the basis of the recently solved structure of native CBL [Clausen, T., et al. (1996) J. Mol. Biol. 262, 202-224], it was possible to elucidate the X-ray structure of the CBLAVG complex and to refine it to an R-factor of 16.4% at 2.2 A resolution. The refined structure reveals the geometry of the bound inhibitor and its interactions with residues in the active site of CBL. Both the X-ray structure and the absorbance spectrum of the CBLAVG complex are compatible with a ketimine as the reaction product. Thus, the inhibitor seems to bind in a similar way to CBL as the substrate, but after alpha-proton abstraction, the reaction proceeds in a CBL nontypical manner, i.e. protonation of PLP-C4', resulting in the "dead-end" ketimine PLP derivative. The CBLAVG structure furthermore suggests a binding mode for rhizobitoxine and explains the failure of MVG to inhibit CBL.

About this StructureAbout this Structure

1CL2 is a Single protein structure of sequence from Escherichia coli with as ligand. Active as Cystathionine beta-lyase, with EC number 4.4.1.8 Full crystallographic information is available from OCA.

ReferenceReference

Slow-binding inhibition of Escherichia coli cystathionine beta-lyase by L-aminoethoxyvinylglycine: a kinetic and X-ray study., Clausen T, Huber R, Messerschmidt A, Pohlenz HD, Laber B, Biochemistry. 1997 Oct 14;36(41):12633-43. PMID:9376370

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-[[Image:1cl2.gif|left|200px]]<br /><applet load="1cl2" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1cl2.gif|left|200px]]<br /><applet load="1cl2" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1cl2, resolution 2.2&Aring;" />
 '''CYSTATHIONINE BETA-LYASE (CBL) FROM ESCHERICHIA COLI IN COMPLEX WITH AMINOETHOXYVINYLGLYCINE'''<br />
 ==Overview==
-The pyridoxal 5'-phosphate (PLP)-dependent cystathionine beta-lyase (CBL), was previously found to be inhibited by the natural toxins rhizobitoxine, and l-aminoethoxyvinylglycine (AVG). The present study characterizes the, interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG), by a combination of kinetic methods and X-ray crystallography. Upon AVG, treatment, time-dependent, slow-binding inhibition [Morrison, J. F. (1982), Trends Biochem. Sci. 7, 102-105] was observed due to the generation of a, long-lived, slowly dissociating enzyme-inhibitor complex. Kinetic analysis, revealed a one-step inhibition mechanism (CBL + AVG --&gt; CBLAVG, Ki = 1.1, +/- 0.3 microM) with an association rate constant (k1) of 336 +/- 40 M-1, s-1. This value is several orders of magnitude lower than typical, bimolecular rate constants of ES formation, suggesting that additional, steps occur before formation of the first detectable CBLAVG complex. Loss, of activity is paralleled by the conversion of the pyridoxaldimine 426 nm, chromophore to a 341 nm-absorbing species. On the basis of the recently, solved structure of native CBL [Clausen, T., et al. (1996) J. Mol. Biol., 262, 202-224], it was possible to elucidate the X-ray structure of the, CBLAVG complex and to refine it to an R-factor of 16.4% at 2.2 A, resolution. The refined structure reveals the geometry of the bound, inhibitor and its interactions with residues in the active site of CBL., Both the X-ray structure and the absorbance spectrum of the CBLAVG complex, are compatible with a ketimine as the reaction product. Thus, the, inhibitor seems to bind in a similar way to CBL as the substrate, but, after alpha-proton abstraction, the reaction proceeds in a CBL nontypical, manner, i.e. protonation of PLP-C4', resulting in the "dead-end" ketimine, PLP derivative. The CBLAVG structure furthermore suggests a binding mode, for rhizobitoxine and explains the failure of MVG to inhibit CBL.
+The pyridoxal 5'-phosphate (PLP)-dependent cystathionine beta-lyase (CBL) was previously found to be inhibited by the natural toxins rhizobitoxine and l-aminoethoxyvinylglycine (AVG). The present study characterizes the interaction of Escherichia coli CBL with AVG and methoxyvinylglycine (MVG) by a combination of kinetic methods and X-ray crystallography. Upon AVG treatment, time-dependent, slow-binding inhibition [Morrison, J. F. (1982) Trends Biochem. Sci. 7, 102-105] was observed due to the generation of a long-lived, slowly dissociating enzyme-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (CBL + AVG --&gt; CBLAVG, Ki = 1.1 +/- 0.3 microM) with an association rate constant (k1) of 336 +/- 40 M-1 s-1. This value is several orders of magnitude lower than typical bimolecular rate constants of ES formation, suggesting that additional steps occur before formation of the first detectable CBLAVG complex. Loss of activity is paralleled by the conversion of the pyridoxaldimine 426 nm chromophore to a 341 nm-absorbing species. On the basis of the recently solved structure of native CBL [Clausen, T., et al. (1996) J. Mol. Biol. 262, 202-224], it was possible to elucidate the X-ray structure of the CBLAVG complex and to refine it to an R-factor of 16.4% at 2.2 A resolution. The refined structure reveals the geometry of the bound inhibitor and its interactions with residues in the active site of CBL. Both the X-ray structure and the absorbance spectrum of the CBLAVG complex are compatible with a ketimine as the reaction product. Thus, the inhibitor seems to bind in a similar way to CBL as the substrate, but after alpha-proton abstraction, the reaction proceeds in a CBL nontypical manner, i.e. protonation of PLP-C4', resulting in the "dead-end" ketimine PLP derivative. The CBLAVG structure furthermore suggests a binding mode for rhizobitoxine and explains the failure of MVG to inhibit CBL.
 ==About this Structure==
-CL2 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 PPG as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cystathionine_beta-lyase Cystathionine beta-lyase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.4.1.8 4.4.1.8] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1CL2 OCA].
+CL2 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=PPG:'>PPG</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cystathionine_beta-lyase Cystathionine beta-lyase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.4.1.8 4.4.1.8] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CL2 OCA].
 ==Reference==
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 [[Category: slow-binding inhibition]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 12:33:00 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:07:09 2008''