1bx3: Difference between revisions

Revision as of 13:00, 21 February 2008

EFFECTS OF COMMONLY USED CRYOPROTECTANTS ON GLYCOGEN PHOSPHORYLASE ACTIVITY AND STRUCTURE

OverviewOverview

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

About this StructureAbout this Structure

1BX3 is a Single protein structure of sequence from Oryctolagus cuniculus with as ligand. Active as Phosphorylase, with EC number 2.4.1.1 Full crystallographic information is available from OCA.

ReferenceReference

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure., Tsitsanou KE, Oikonomakos NG, Zographos SE, Skamnaki VT, Gregoriou M, Watson KA, Johnson LN, Fleet GW, Protein Sci. 1999 Apr;8(4):741-9. PMID:10211820

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-[[Image:1bx3.gif|left|200px]]<br /><applet load="1bx3" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1bx3.gif|left|200px]]<br /><applet load="1bx3" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1bx3, resolution 2.30&Aring;" />
 '''EFFECTS OF COMMONLY USED CRYOPROTECTANTS ON GLYCOGEN PHOSPHORYLASE ACTIVITY AND STRUCTURE'''<br />
 ==Overview==
-The effects of a number of cryoprotectants on the kinetic and structural, properties of glycogen phosphorylase b have been investigated. Kinetic, studies showed that glycerol, one of the most commonly used, cryoprotectants in X-ray crystallographic studies, is a competitive, inhibitor with respect to substrate glucose-1-P with an apparent Ki value, of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that, glycerol binds at the catalytic site and competes with glucose analogues, that bind at the catalytic site, thus preventing the formation of, complexes. This necessitated a change in the conditions for cryoprotection, in crystallographic binding experiments with glycogen phosphorylase. It, was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs), of various molecular weights, and dimethyl sulfoxide (DMSO) activated, glycogen phosphorylase b to different extents, by stabilizing its most, active conformation, while sucrose acted as a noncompetitive inhibitor and, ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P., A parallel experimental investigation by X-ray crystallography showed, that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do, not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding, studies with glycogen phosphorylase.
+The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.
 ==About this Structure==
-BX3 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Oryctolagus_cuniculus Oryctolagus cuniculus] with PLP as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Phosphorylase Phosphorylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.1.1 2.4.1.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1BX3 OCA].
+BX3 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Oryctolagus_cuniculus Oryctolagus cuniculus] with <scene name='pdbligand=PLP:'>PLP</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Phosphorylase Phosphorylase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.4.1.1 2.4.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1BX3 OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Phosphorylase]]
 [[Category: Single protein]]
-[[Category: Fleet, G.W.J.]]
+[[Category: Fleet, G W.J.]]
 [[Category: Gregoriou, M.]]
-[[Category: Johnson, L.N.]]
+[[Category: Johnson, L N.]]
-[[Category: Oikonomakos, N.G.]]
+[[Category: Oikonomakos, N G.]]
-[[Category: Skamnaki, V.T.]]
+[[Category: Skamnaki, V T.]]
-[[Category: Tsitsanou, K.E.]]
+[[Category: Tsitsanou, K E.]]
-[[Category: Watson, K.A.]]
+[[Category: Watson, K A.]]
-[[Category: Zographos, S.E.]]
+[[Category: Zographos, S E.]]
 [[Category: PLP]]
 [[Category: binding]]
@@ Line 31: / Line 31: @@
 [[Category: phosphorylase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 11:59:14 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:00:01 2008''