DOPA decarboxylase - Revision history

Michal Harel at 06:47, 8 July 2021

2021-07-08T06:47:29Z

←Older revision		Revision as of 06:47, 8 July 2021
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	[[image:dopa.png\|thumb\|left\|300px\|'''Dopamine Synthesis''']]		[[image:dopa.png\|thumb\|left\|300px\|'''Dopamine Synthesis''']]
	{{Clear}}		{{Clear}}
-	'''DOPA decarboxylase''' (DDC, aromatic L-amino acid decarboxylase, tryptophan decarboxylase, 5-hydroxytryptophan decarboxylase, AAAD) ([[EC Number\|EC]] 4.1.1.28) is an approximately 104 kDa protein that belongs to the '''aspartate aminotransferase family''' (fold type 1) of '''[http://en.wikipedia.org/wiki/Pyridoxal_phosphate ''PLP'']-dependent''' (vitamin B6-dependent) enzymes. The catalytically active form of the enzyme exists as a homodimer, typical of this class of enzymes.<ref name="schneider">PMID:10673430 </ref> The homodimeric form of the enzyme purified from [http://en.wikipedia.org/wiki/Sus_scrofa ''sus scrofa''] is shown in complex with the inhibitor '''[http://en.wikipedia.org/wiki/Carbidopa ''carbidopa'']''' to the right.	+	'''DOPA decarboxylase''' ('''DDC, aromatic L-amino acid decarboxylase, tryptophan decarboxylase, 5-hydroxytryptophan decarboxylase, AAAD''') ([[EC Number\|EC]] 4.1.1.28) is an approximately 104 kDa protein that belongs to the '''aspartate aminotransferase family''' (fold type 1) of '''[http://en.wikipedia.org/wiki/Pyridoxal_phosphate ''PLP'']-dependent''' (vitamin B6-dependent) enzymes. The catalytically active form of the enzyme exists as a homodimer, typical of this class of enzymes.<ref name="schneider">PMID:10673430 </ref> The homodimeric form of the enzyme purified from [http://en.wikipedia.org/wiki/Sus_scrofa ''sus scrofa''] is shown in complex with the inhibitor '''[http://en.wikipedia.org/wiki/Carbidopa ''carbidopa'']''' to the right.

	DDC catalyzes the conversion of aromatic amino acids into their corresponding amines. DOPA decarboxylase is responsible for the synthesis of '''[http://en.wikipedia.org/wiki/Dopamine ''dopamine'']''' and [http://en.wikipedia.org/wiki/Serotoninn ''serotonin''] from '''[http://en.wikipedia.org/wiki/L-dopa ''L-DOPA'']''' and [http://en.wikipedia.org/wiki/L-5-Hydroxytryptophan ''L-5-hydroxytryptophan''], respectively. It is highly stereospecific, yet relatively nonspecific in terms of substrate, making it a somewhat uninteresting enzyme to study. Although it is not typically a rate-determining step of dopamine synthesis, the decarboxylation of L-DOPA to dopamine by DDC is the controlling step for individuals with '''[http://en.wikipedia.org/wiki/Parkinson%27s_disease ''Parkinson's disease'']'''<ref name="hadjiiconstantinou">PMID:1904055 </ref> , the second most common neurodegenerative disorder, occuring in 1% of the population over the age of 65. The loss of dopaminergic neurons is the main cause of cognitive impairment and tremors observed in patients with the disease. The hallmark of the disease is the formation of [http://en.wikipedia.org/wiki/alpha-synuclein ''alpha-synuclein''] containing [http://en.wikipedia.org/wiki/Lewy_body ''Lewy bodies''].		DDC catalyzes the conversion of aromatic amino acids into their corresponding amines. DOPA decarboxylase is responsible for the synthesis of '''[http://en.wikipedia.org/wiki/Dopamine ''dopamine'']''' and [http://en.wikipedia.org/wiki/Serotoninn ''serotonin''] from '''[http://en.wikipedia.org/wiki/L-dopa ''L-DOPA'']''' and [http://en.wikipedia.org/wiki/L-5-Hydroxytryptophan ''L-5-hydroxytryptophan''], respectively. It is highly stereospecific, yet relatively nonspecific in terms of substrate, making it a somewhat uninteresting enzyme to study. Although it is not typically a rate-determining step of dopamine synthesis, the decarboxylation of L-DOPA to dopamine by DDC is the controlling step for individuals with '''[http://en.wikipedia.org/wiki/Parkinson%27s_disease ''Parkinson's disease'']'''<ref name="hadjiiconstantinou">PMID:1904055 </ref> , the second most common neurodegenerative disorder, occuring in 1% of the population over the age of 65. The loss of dopaminergic neurons is the main cause of cognitive impairment and tremors observed in patients with the disease. The hallmark of the disease is the formation of [http://en.wikipedia.org/wiki/alpha-synuclein ''alpha-synuclein''] containing [http://en.wikipedia.org/wiki/Lewy_body ''Lewy bodies''].
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	{{Clear}}		{{Clear}}
	This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.		This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.
-
-
-
-
-

	===Mechanism Breakdown===		===Mechanism Breakdown===
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	With it's possible relation to degenerative dopamine-producing cells in the brain, administration of L-DOPA can increase the amount of synthesized dopamine in the nerve cell; whereas, direct treatment with dopamine is not sufficient as dopamine itself cannot pass the blood-brain barrier<ref name=Burkhard>PMID: 11685243 </ref>. Even still, only a small percentage of the dose actually reaches the nervous system, with the remaining majority being rapidly decarboxylated to dopamine in the blood stream. This dopamine-rich blood causes side effects of nausea, daytime sleepiness, orthostatic hypotension, involuntary movements, decreased appetite, insomnia, and cramping. Addition of a DDC inhibitor would block peripheral conversion to dopamine and allow a greater percentage of L-DOPA to reach the brain, causing an increase in brain dopamine levels, and reducing the side effects of dopamine-rich blood.		With it's possible relation to degenerative dopamine-producing cells in the brain, administration of L-DOPA can increase the amount of synthesized dopamine in the nerve cell; whereas, direct treatment with dopamine is not sufficient as dopamine itself cannot pass the blood-brain barrier<ref name=Burkhard>PMID: 11685243 </ref>. Even still, only a small percentage of the dose actually reaches the nervous system, with the remaining majority being rapidly decarboxylated to dopamine in the blood stream. This dopamine-rich blood causes side effects of nausea, daytime sleepiness, orthostatic hypotension, involuntary movements, decreased appetite, insomnia, and cramping. Addition of a DDC inhibitor would block peripheral conversion to dopamine and allow a greater percentage of L-DOPA to reach the brain, causing an increase in brain dopamine levels, and reducing the side effects of dopamine-rich blood.

-
-
	==Classification==		==Classification==
	----		----
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	[[6eew]] – MpDDC + Trp – Madagascar periwinkle<br />		[[6eew]] – MpDDC + Trp – Madagascar periwinkle<br />
	[[6eem]] – MpDDC + Tyr<br />		[[6eem]] – MpDDC + Tyr<br />
		+	[[6liu]] – poDDC - poppy<br />
		+	[[6liv]] – poDDC + PLP derivative<br />

	==References==		==References==

Michal Harel at 09:15, 12 June 2019

2019-06-12T09:15:34Z

←Older revision		Revision as of 09:15, 12 June 2019
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	Updated on {{REVISIONDAY2}}-{{MONTHNAME\|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}		Updated on {{REVISIONDAY2}}-{{MONTHNAME\|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}

		+	[[3rbf]], [[3rbl]] – hDDC – human<br />
		+	[[3rch]] – hDDC + vitamin B6 phosphate + pyridoxal phosphate
	[[3k40]] – DDC – ''Drosophila melanogaster''<br />		[[3k40]] – DDC – ''Drosophila melanogaster''<br />
	[[1js3]] – pDDC + inhibitor – pig<br />		[[1js3]] – pDDC + inhibitor – pig<br />
	[[1js6]] - pDDC<br />		[[1js6]] - pDDC<br />
-	[[~~3rbf]], [[3rbl~~]] – ~~hDDC~~ – ~~human~~<br />	+	[[6eew]] – MpDDC + Trp – Madagascar periwinkle<br />
-	[[~~3rch~~]] – ~~hDDC~~ + ~~vitamin B6 phosphate + pyridoxal phosphate~~	+	[[6eem]] – MpDDC + Tyr<br />

	==References==		==References==

Alexander Berchansky at 12:36, 3 September 2015

2015-09-03T12:36:50Z

←Older revision		Revision as of 12:36, 3 September 2015
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	{{Clear}}		{{Clear}}
	In the resting state, PLP forms a covalent bond with the amino group of the active site Lysine (internal aldimine). Upon introduction of the substrate into the active site, a new Schiff base is generated (external aldimine). This transimination step is common to '''all''' PLP-dependent enzymes.		In the resting state, PLP forms a covalent bond with the amino group of the active site Lysine (internal aldimine). Upon introduction of the substrate into the active site, a new Schiff base is generated (external aldimine). This transimination step is common to '''all''' PLP-dependent enzymes.
-	Although these enzymes have wide range of function, they can be classified into only five structural families: the '''aspartate amino transferase''', the tryptophan synthase β, the alanine racemase, the D-amino acid, and the glycogen phosphorylase. <ref name="percudani">PMID:12949584 </ref> <ref name="schneider">PMID:10673430 </ref> [[image:plp6.jpg\|thumb\|left\|400px\|'''The PLP is bound covalently to lysine residues in a Schiff base linkage (aldimine). In this form, it reacts with many free amino acids to replace the Schiff base to the lysine of the enzyme with a Schiff base to the amino acid substrate.'']]	+	Although these enzymes have wide range of function, they can be classified into only five structural families: the '''aspartate amino transferase''', the tryptophan synthase β, the alanine racemase, the D-amino acid, and the glycogen phosphorylase. <ref name="percudani">PMID:12949584 </ref> <ref name="schneider">PMID:10673430 </ref> [[image:plp6.jpg\|thumb\|left\|400px\|'''The PLP is bound covalently to lysine residues in a Schiff base linkage (aldimine). In this form, it reacts with many free amino acids to replace the Schiff base to the lysine of the enzyme with a Schiff base to the amino acid substrate.'']]
		+	{{Clear}}
	----		----
	===The Aspartate Aminotransferase Family===		===The Aspartate Aminotransferase Family===
	[[image:aspartateamino.png\|thumb\|left\|200px\|'''Aspartate Aminotransferase''']]		[[image:aspartateamino.png\|thumb\|left\|200px\|'''Aspartate Aminotransferase''']]
		+	{{Clear}}
	This family of PLP-dependent enzymes is also referred to as '''fold-type I''', with aspartate aminotransferase serving as the prototype. It is the most common structure of the five classes of PLP-dependent enzymes. This fold it is found in a variety of aminotransferases and decarboxylases, amongst them '''DOPA decarboxylase'''. PLP-dependent enzymes belonging to this family are catalytically active as homodimers and share a common, well-characterized structure, despite low-sequence identity. Each subunit has a large domain and a small domain. The central feature of the large domain is a seven-stranded β sheet. The small domain has either a three or four-stranded β sheet that is surrounded by α helices on one side. The cofactor PLP is covalently attached to a lysine residue in the large domain and is anchored in a way that allows the aromatic ring of PLP to pack against neighboring β strands. The active site is located in a cleft between the two domains at the interface between the two subunits. '''''Thus, enzymes of fold-type I have residues from both domains and both subunits involved in PLP-binding.'''''		This family of PLP-dependent enzymes is also referred to as '''fold-type I''', with aspartate aminotransferase serving as the prototype. It is the most common structure of the five classes of PLP-dependent enzymes. This fold it is found in a variety of aminotransferases and decarboxylases, amongst them '''DOPA decarboxylase'''. PLP-dependent enzymes belonging to this family are catalytically active as homodimers and share a common, well-characterized structure, despite low-sequence identity. Each subunit has a large domain and a small domain. The central feature of the large domain is a seven-stranded β sheet. The small domain has either a three or four-stranded β sheet that is surrounded by α helices on one side. The cofactor PLP is covalently attached to a lysine residue in the large domain and is anchored in a way that allows the aromatic ring of PLP to pack against neighboring β strands. The active site is located in a cleft between the two domains at the interface between the two subunits. '''''Thus, enzymes of fold-type I have residues from both domains and both subunits involved in PLP-binding.'''''
	----		----
	[[image:dopadecarb.png\|thumb\|200px\|'''DOPA Decarboxylase''']][[image:super1.png\|thumb\|200px\|'''DOPA decarboxylase superimposed on aspartate aminotransferase''']]		[[image:dopadecarb.png\|thumb\|200px\|'''DOPA Decarboxylase''']][[image:super1.png\|thumb\|200px\|'''DOPA decarboxylase superimposed on aspartate aminotransferase''']]
		+	{{Clear}}
	----		----
	===DOPA Decarboxylase===		===DOPA Decarboxylase===
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	----		----
	As well, a '''salt bridge''' exists between the carboxyl group of <scene name='DOPA_decarboxylase/Aspartic/1'>Asp271</scene> and the protonated pyridine nitrogen of PLP to further stabilize intermediate. Essentially, a salt bridge combines hydrogen bonding and electrostatic interactions (two common types non-covalent interactions). This interaction serves to provide an electron sink that can stabilize the carbanionic intermediates <ref name="jansonius">PMID:9914259 </ref> . PLP is further anchored to the protein by an extended '''hydrogen bond network''', as shown below.		As well, a '''salt bridge''' exists between the carboxyl group of <scene name='DOPA_decarboxylase/Aspartic/1'>Asp271</scene> and the protonated pyridine nitrogen of PLP to further stabilize intermediate. Essentially, a salt bridge combines hydrogen bonding and electrostatic interactions (two common types non-covalent interactions). This interaction serves to provide an electron sink that can stabilize the carbanionic intermediates <ref name="jansonius">PMID:9914259 </ref> . PLP is further anchored to the protein by an extended '''hydrogen bond network''', as shown below.
-	[[image:h-bonding.png\|thumb\|center\|300px\|'''H-bonding network of PLP in the active site''']]	+	[[image:h-bonding.png\|thumb\|center\|300px\|'''H-bonding network of PLP in the active site''']]
		+	{{Clear}}
	----		----
	The only two active site residues from the adjacent monomer, Ile-101 and Phe-103, are part of the substrate binding pocket.		The only two active site residues from the adjacent monomer, Ile-101 and Phe-103, are part of the substrate binding pocket.
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	===Inhibitor Binding===		===Inhibitor Binding===
	[[image:carbiDOPA.png\|thumb\|left\|200px\|'''carbiDOPA''']]		[[image:carbiDOPA.png\|thumb\|left\|200px\|'''carbiDOPA''']]
		+	{{Clear}}
	The inhibitor <scene name='DOPA_decarboxylase/Carbidopa/1'>carbiDOPA</scene> binds to the enzyme by forming a hydrazone linkage with PLP through its hydrazine moiety. The catechol ring of carbiDOPA is deeply buried in the active site cleft and is stabilized by <scene name='DOPA_decarboxylase/Vanderwaals/1'>van der waals contact</scene> with Ile-101 and Phe-103. The 4' hydroxyl group of the catechol ring participates in hydrogen bonding with <scene name='DOPA_decarboxylase/Thr-82/1'>Thr-82</scene>, further stabilizing the inhibitor in the active site cleft. PLP is further involved in substrate binding by forming a hydrogen bond to the 3' of the catechol ring. <scene name='DOPA_decarboxylase/His192/1'>His-192</scene>, a highly conserved residue of PLP-dependent decarboxylases <ref name="ishii">PMID:8889823 </ref> hydrogen bonds to the carboxylate group of carbiDOPA.		The inhibitor <scene name='DOPA_decarboxylase/Carbidopa/1'>carbiDOPA</scene> binds to the enzyme by forming a hydrazone linkage with PLP through its hydrazine moiety. The catechol ring of carbiDOPA is deeply buried in the active site cleft and is stabilized by <scene name='DOPA_decarboxylase/Vanderwaals/1'>van der waals contact</scene> with Ile-101 and Phe-103. The 4' hydroxyl group of the catechol ring participates in hydrogen bonding with <scene name='DOPA_decarboxylase/Thr-82/1'>Thr-82</scene>, further stabilizing the inhibitor in the active site cleft. PLP is further involved in substrate binding by forming a hydrogen bond to the 3' of the catechol ring. <scene name='DOPA_decarboxylase/His192/1'>His-192</scene>, a highly conserved residue of PLP-dependent decarboxylases <ref name="ishii">PMID:8889823 </ref> hydrogen bonds to the carboxylate group of carbiDOPA.
	[[image:actsite.png\|thumb\|center\|300px\|'''Key interactions between the active site residues, PLP, and carbiDOPA''']]		[[image:actsite.png\|thumb\|center\|300px\|'''Key interactions between the active site residues, PLP, and carbiDOPA''']]
-		+	{{Clear}}
	'''Other inhibitors''' include [http://en.wikipedia.org/wiki/Benserazide Benserazide] (Serazide), α-Difluoromethyl-DOPA, and α-methyldopa. However, Benserazide is unapproved for use in the U.S. and is replaced by carbiDOPA instead. Both inhibitors act by preventing peripheral metabolism of L-DOPA, and cannot cross the blood-brain barrier.		'''Other inhibitors''' include [http://en.wikipedia.org/wiki/Benserazide Benserazide] (Serazide), α-Difluoromethyl-DOPA, and α-methyldopa. However, Benserazide is unapproved for use in the U.S. and is replaced by carbiDOPA instead. Both inhibitors act by preventing peripheral metabolism of L-DOPA, and cannot cross the blood-brain barrier.

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	{{Clear}}		{{Clear}}
	Once again, the transimination step (conversion of internal to external aldimine) is common to all PLP-dependent enzymes. The unique absorption wavelengths of the PLP intermediates has allowed for their straightforward detection (for example, an internal Schiff base in the enolimine form absorbs at 310-330 nm, whereas the quinonoid intermediate absorbs at ~500 nm). The orientation of the quinonoid intermediate allows for stereospecific decarboxylation of the substrate at the alpha carbon, as predicted by '''Dunathan's stereoelectronic hypothesis''' <ref name="dunathan">PMID:224217 </ref>, in which he proposed that the substrate binds PLP such that the external aldimine intermediate is oriented perpendicular to the coenzyme pi bonding system. In doing so, the sigma-pi orbital overlap in the transition state is maximized, thus maximizing the rate of the reaction.		Once again, the transimination step (conversion of internal to external aldimine) is common to all PLP-dependent enzymes. The unique absorption wavelengths of the PLP intermediates has allowed for their straightforward detection (for example, an internal Schiff base in the enolimine form absorbs at 310-330 nm, whereas the quinonoid intermediate absorbs at ~500 nm). The orientation of the quinonoid intermediate allows for stereospecific decarboxylation of the substrate at the alpha carbon, as predicted by '''Dunathan's stereoelectronic hypothesis''' <ref name="dunathan">PMID:224217 </ref>, in which he proposed that the substrate binds PLP such that the external aldimine intermediate is oriented perpendicular to the coenzyme pi bonding system. In doing so, the sigma-pi orbital overlap in the transition state is maximized, thus maximizing the rate of the reaction.
-	[[image:Dunathan.png\|thumb\|left\|300px\|'''Dunathan's Stereoeletronic Hypothesis, 1966''']]	+	[[image:Dunathan.png\|thumb\|left\|300px\|'''Dunathan's Stereoeletronic Hypothesis, 1966''']]
		+	{{Clear}}
	This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.		This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.

Alexander Berchansky at 14:45, 23 December 2013

2013-12-23T14:45:20Z

(Difference between revisions)

Michal Harel at 11:57, 7 March 2013

2013-03-07T11:57:53Z

←Older revision		Revision as of 11:57, 7 March 2013
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	==3D structures of DOPA decarboxylase==		==3D structures of DOPA decarboxylase==

-	~~''Update November 2011''~~	+	Updated on {{REVISIONDAY2}}-{{MONTHNAME\|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}

	[[3k40]] – DDC – ''Drosophila melanogaster''<br />		[[3k40]] – DDC – ''Drosophila melanogaster''<br />

Brian Hernandez: AddAuthor

2012-07-10T09:40:51Z

AddAuthor

←Older revision

Revision as of 09:40, 10 July 2012

Michal Harel at 08:54, 10 July 2012

2012-07-10T08:54:15Z

←Older revision		Revision as of 08:54, 10 July 2012
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	The mechanism of DDC catalyzed decarboxylation of L-Dopa to Dopamine has been well-studied due to the enzymes role in PD. Shown below is the mechanism of decarboxylation, as determined by several experimental approaches, including site-directed mutagenesis, UV-Vis Spectroscopy, X-Ray Crystallography, Multiple Sequence Alignment, and Stopped-Flow Spectroscopy. [[image:slide1.png\|thumb\|center\|600px\|]] Once again, the transimination step (conversion of internal to external aldimine) is common to all PLP-dependent enzymes. The unique absorption wavelengths of the PLP intermediates has allowed for their straightforward detection (for example, an internal Schiff base in the enolimine form absorbs at 310-330 nm, whereas the quinonoid intermediate absorbs at ~500 nm). The orientation of the quinonoid intermediate allows for stereospecific decarboxylation of the substrate at the alpha carbon, as predicted by '''Dunathan's stereoelectronic hypothesis''' <ref name="dunathan">PMID:224217 </ref>, in which he proposed that the substrate binds PLP such that the external aldimine intermediate is oriented perpendicular to the coenzyme pi bonding system. In doing so, the sigma-pi orbital overlap in the transition state is maximized, thus maximizing the rate of the reaction.		The mechanism of DDC catalyzed decarboxylation of L-Dopa to Dopamine has been well-studied due to the enzymes role in PD. Shown below is the mechanism of decarboxylation, as determined by several experimental approaches, including site-directed mutagenesis, UV-Vis Spectroscopy, X-Ray Crystallography, Multiple Sequence Alignment, and Stopped-Flow Spectroscopy. [[image:slide1.png\|thumb\|center\|600px\|]] Once again, the transimination step (conversion of internal to external aldimine) is common to all PLP-dependent enzymes. The unique absorption wavelengths of the PLP intermediates has allowed for their straightforward detection (for example, an internal Schiff base in the enolimine form absorbs at 310-330 nm, whereas the quinonoid intermediate absorbs at ~500 nm). The orientation of the quinonoid intermediate allows for stereospecific decarboxylation of the substrate at the alpha carbon, as predicted by '''Dunathan's stereoelectronic hypothesis''' <ref name="dunathan">PMID:224217 </ref>, in which he proposed that the substrate binds PLP such that the external aldimine intermediate is oriented perpendicular to the coenzyme pi bonding system. In doing so, the sigma-pi orbital overlap in the transition state is maximized, thus maximizing the rate of the reaction.
	[[image:Dunathan.png\|thumb\|left\|300px\|'''Dunathan's Stereoeletronic Hypothesis, 1966''']] This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.		[[image:Dunathan.png\|thumb\|left\|300px\|'''Dunathan's Stereoeletronic Hypothesis, 1966''']] This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.
		+
		+
		+
		+
		+

	===Mechanism Breakdown===		===Mechanism Breakdown===

Michal Harel at 08:48, 10 July 2012

2012-07-10T08:48:10Z

←Older revision		Revision as of 08:48, 10 July 2012
Line 46:		Line 46:
	[[image:carbiDOPA.png\|thumb\|left\|200px\|'''carbiDOPA''']]The inhibitor <scene name='DOPA_decarboxylase/Carbidopa/1'>carbiDOPA</scene> binds to the enzyme by forming a hydrazone linkage with PLP through its hydrazine moiety. The catechol ring of carbiDOPA is deeply buried in the active site cleft and is stabilized by <scene name='DOPA_decarboxylase/Vanderwaals/1'>van der waals contact</scene> with Ile-101 and Phe-103. The 4' hydroxyl group of the catechol ring participates in hydrogen bonding with <scene name='DOPA_decarboxylase/Thr-82/1'>Thr-82</scene>, further stabilizing the inhibitor in the active site cleft. PLP is further involved in substrate binding by forming a hydrogen bond to the 3' of the catechol ring. <scene name='DOPA_decarboxylase/His192/1'>His-192</scene>, a highly conserved residue of PLP-dependent decarboxylases <ref name="ishii">PMID:8889823 </ref> hydrogen bonds to the carboxylate group of carbiDOPA.		[[image:carbiDOPA.png\|thumb\|left\|200px\|'''carbiDOPA''']]The inhibitor <scene name='DOPA_decarboxylase/Carbidopa/1'>carbiDOPA</scene> binds to the enzyme by forming a hydrazone linkage with PLP through its hydrazine moiety. The catechol ring of carbiDOPA is deeply buried in the active site cleft and is stabilized by <scene name='DOPA_decarboxylase/Vanderwaals/1'>van der waals contact</scene> with Ile-101 and Phe-103. The 4' hydroxyl group of the catechol ring participates in hydrogen bonding with <scene name='DOPA_decarboxylase/Thr-82/1'>Thr-82</scene>, further stabilizing the inhibitor in the active site cleft. PLP is further involved in substrate binding by forming a hydrogen bond to the 3' of the catechol ring. <scene name='DOPA_decarboxylase/His192/1'>His-192</scene>, a highly conserved residue of PLP-dependent decarboxylases <ref name="ishii">PMID:8889823 </ref> hydrogen bonds to the carboxylate group of carbiDOPA.
	[[image:actsite.png\|thumb\|center\|300px\|'''Key interactions between the active site residues, PLP, and carbiDOPA''']]		[[image:actsite.png\|thumb\|center\|300px\|'''Key interactions between the active site residues, PLP, and carbiDOPA''']]
		+
		+	'''Other inhibitors''' include [http://en.wikipedia.org/wiki/Benserazide Benserazide] (Serazide), α-Difluoromethyl-DOPA, and α-methyldopa. However, Benserazide is unapproved for use in the U.S. and is replaced by carbiDOPA instead. Both inhibitors act by preventing peripheral metabolism of L-DOPA, and cannot cross the blood-brain barrier.
		+
	----		----
	===Flexible Loop===		===Flexible Loop===
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	====Step 5: Formation of an Internal Aldimine and Product Release====		====Step 5: Formation of an Internal Aldimine and Product Release====
	Formation of an internal aldimine between PLP and Lys303 regenerates the enzyme. It has been shown that Lys303 plays a role in product release by presumably displacing the amino group of the product by nucleophilic attack of the imine bond of the external aldimine.		Formation of an internal aldimine between PLP and Lys303 regenerates the enzyme. It has been shown that Lys303 plays a role in product release by presumably displacing the amino group of the product by nucleophilic attack of the imine bond of the external aldimine.
		+
		+	==DDC and Parkinson's Disease==
		+	==='''Treatment'''===
		+	Parkinson's disease can be characterized by [http://en.wikipedia.org/wiki/Tremor tremor], [http://en.wikipedia.org/wiki/Bradykinesia#Bradykinesia bradykinesia], rigidity, and postural instability. [[Image:dopamine levels.jpeg\|thmb\|right\|300px\|'''Dopamine Levels''']]With it's possible relation to degenerative dopamine-producing cells in the brain, administration of L-DOPA can increase the amount of synthesized dopamine in the nerve cell; whereas, direct treatment with dopamine is not sufficient as dopamine itself cannot pass the blood-brain barrier<ref name=Burkhard>PMID: 11685243 </ref>. Even still, only a small percentage of the dose actually reaches the nervous system, with the remaining majority being rapidly decarboxylated to dopamine in the blood stream. This dopamine-rich blood causes side effects of nausea, daytime sleepiness, orthostatic hypotension, involuntary movements, decreased appetite, insomnia, and cramping. Addition of a DDC inhibitor would block peripheral conversion to dopamine and allow a greater percentage of L-DOPA to reach the brain, causing an increase in brain dopamine levels, and reducing the side effects of dopamine-rich blood.
		+

Brittany Todd at 16:10, 1 May 2012

2012-05-01T16:10:46Z

←Older revision		Revision as of 16:10, 1 May 2012
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	====Step 3: Formation of a Quinonoid Intermediate====		====Step 3: Formation of a Quinonoid Intermediate====
	The formation of the quinonoid intermediate is common to all PLP-dependent enzymes, yet the orientation of the intermediate, as determined by key residues of the enzyme active site, determines the subsequent reaction (for example whether it will be a decarboxylation or transamination). A salt bridge that exists between Asp271 and the protonated pyridine nitrogen of PLP further enhances the ability of PLP to act as an electron sink and promote catalysis. As well, During the formation of the quinonoid intermediate, carbon dioxide is released.		The formation of the quinonoid intermediate is common to all PLP-dependent enzymes, yet the orientation of the intermediate, as determined by key residues of the enzyme active site, determines the subsequent reaction (for example whether it will be a decarboxylation or transamination). A salt bridge that exists between Asp271 and the protonated pyridine nitrogen of PLP further enhances the ability of PLP to act as an electron sink and promote catalysis. As well, During the formation of the quinonoid intermediate, carbon dioxide is released.
-	====Step 4: Formation of an External Aldimine====	+	====Step 4: Formation of an External Aldimine====
	The formation of the external aldimine between the product and PLP is the fourth step of the reaction. Here, Tyr332, with the assistance of His192, likely donates a proton to the quinonoid Cα intermediate.		The formation of the external aldimine between the product and PLP is the fourth step of the reaction. Here, Tyr332, with the assistance of His192, likely donates a proton to the quinonoid Cα intermediate.
	====Step 5: Formation of an Internal Aldimine and Product Release====		====Step 5: Formation of an Internal Aldimine and Product Release====

Brittany Todd at 16:09, 1 May 2012

2012-05-01T16:09:56Z

←Older revision		Revision as of 16:09, 1 May 2012
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	The mechanism of DDC catalyzed decarboxylation of L-Dopa to Dopamine has been well-studied due to the enzymes role in PD. Shown below is the mechanism of decarboxylation, as determined by several experimental approaches, including site-directed mutagenesis, UV-Vis Spectroscopy, X-Ray Crystallography, Multiple Sequence Alignment, and Stopped-Flow Spectroscopy. [[image:slide1.png\|thumb\|center\|600px\|]] Once again, the transimination step (conversion of internal to external aldimine) is common to all PLP-dependent enzymes. The unique absorption wavelengths of the PLP intermediates has allowed for their straightforward detection (for example, an internal Schiff base in the enolimine form absorbs at 310-330 nm, whereas the quinonoid intermediate absorbs at ~500 nm). The orientation of the quinonoid intermediate allows for stereospecific decarboxylation of the substrate at the alpha carbon, as predicted by '''Dunathan's stereoelectronic hypothesis''' <ref name="dunathan">PMID:224217 </ref>, in which he proposed that the substrate binds PLP such that the external aldimine intermediate is oriented perpendicular to the coenzyme pi bonding system. In doing so, the sigma-pi orbital overlap in the transition state is maximized, thus maximizing the rate of the reaction.		The mechanism of DDC catalyzed decarboxylation of L-Dopa to Dopamine has been well-studied due to the enzymes role in PD. Shown below is the mechanism of decarboxylation, as determined by several experimental approaches, including site-directed mutagenesis, UV-Vis Spectroscopy, X-Ray Crystallography, Multiple Sequence Alignment, and Stopped-Flow Spectroscopy. [[image:slide1.png\|thumb\|center\|600px\|]] Once again, the transimination step (conversion of internal to external aldimine) is common to all PLP-dependent enzymes. The unique absorption wavelengths of the PLP intermediates has allowed for their straightforward detection (for example, an internal Schiff base in the enolimine form absorbs at 310-330 nm, whereas the quinonoid intermediate absorbs at ~500 nm). The orientation of the quinonoid intermediate allows for stereospecific decarboxylation of the substrate at the alpha carbon, as predicted by '''Dunathan's stereoelectronic hypothesis''' <ref name="dunathan">PMID:224217 </ref>, in which he proposed that the substrate binds PLP such that the external aldimine intermediate is oriented perpendicular to the coenzyme pi bonding system. In doing so, the sigma-pi orbital overlap in the transition state is maximized, thus maximizing the rate of the reaction.
	[[image:Dunathan.png\|thumb\|left\|300px\|'''Dunathan's Stereoeletronic Hypothesis, 1966''']] This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.		[[image:Dunathan.png\|thumb\|left\|300px\|'''Dunathan's Stereoeletronic Hypothesis, 1966''']] This way, the developing p orbital is aligned for maximal overlap with the extended p system, lowering the energy of the transition state and increasing the rate of the reaction. As well, by controlling substrate orientation, the enzyme can distinguish between '''deprotonation''' and '''decarboxylation'''.
-	[[image:untitled.png\|thumb\|center\|400px\|]]

	===Mechanism Breakdown===		===Mechanism Breakdown===
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	The first step of the reaction involves the binding of PLP to the enzyme via a Schiff base linkage between the aldehyde group of PLP and the ε—amino group of Lys303. An extensive hydrogen bond network further anchors PLP to the enzyme.		The first step of the reaction involves the binding of PLP to the enzyme via a Schiff base linkage between the aldehyde group of PLP and the ε—amino group of Lys303. An extensive hydrogen bond network further anchors PLP to the enzyme.
	====Step 2: Formation of an External Aldimine====		====Step 2: Formation of an External Aldimine====
-	The second step of the reaction involves the binding of PLP with the substrate via a Schiff base linkage. The imine formed in the first step of the reaction is more succeptible than a free aldehyde to nucleophilic attack by the amino group of the substrate. Thus, not only does the internal aldimine between PLP and the Lys303 bind cofactor, it also serves to facilitate chemistry occurring in this second step.	+	The second step of the reaction involves the binding of PLP with the substrate via a Schiff base linkage. The imine formed in the first step of the reaction is more succeptible than a free aldehyde to nucleophilic attack by the amino group of the substrate. Thus, not only does the internal aldimine between PLP and the Lys303 bind cofactor, it also serves to facilitate chemistry occurring in this second step.[[image:untitled.png\|thumb\|center\|400px\|]]
	====Step 3: Formation of a Quinonoid Intermediate====		====Step 3: Formation of a Quinonoid Intermediate====
	The formation of the quinonoid intermediate is common to all PLP-dependent enzymes, yet the orientation of the intermediate, as determined by key residues of the enzyme active site, determines the subsequent reaction (for example whether it will be a decarboxylation or transamination). A salt bridge that exists between Asp271 and the protonated pyridine nitrogen of PLP further enhances the ability of PLP to act as an electron sink and promote catalysis. As well, During the formation of the quinonoid intermediate, carbon dioxide is released.		The formation of the quinonoid intermediate is common to all PLP-dependent enzymes, yet the orientation of the intermediate, as determined by key residues of the enzyme active site, determines the subsequent reaction (for example whether it will be a decarboxylation or transamination). A salt bridge that exists between Asp271 and the protonated pyridine nitrogen of PLP further enhances the ability of PLP to act as an electron sink and promote catalysis. As well, During the formation of the quinonoid intermediate, carbon dioxide is released.
-	====Step 4: Formation of an External Aldimine===	+	====Step 4: Formation of an External Aldimine====
	The formation of the external aldimine between the product and PLP is the fourth step of the reaction. Here, Tyr332, with the assistance of His192, likely donates a proton to the quinonoid Cα intermediate.		The formation of the external aldimine between the product and PLP is the fourth step of the reaction. Here, Tyr332, with the assistance of His192, likely donates a proton to the quinonoid Cα intermediate.
	====Step 5: Formation of an Internal Aldimine and Product Release====		====Step 5: Formation of an Internal Aldimine and Product Release====