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4350-09-8 Usage

Chemical Properties

white to pale grey powder

Uses

Different sources of media describe the Uses of 4350-09-8 differently. You can refer to the following data:
1. 5-Hydroxy-L-Tryptophan is a hydroxylated metabolite of L-Tryptophan
2. 5-Hydroxy-L-tryptophan has been used:to inject experimental mice for serotonin detectionas a precursor of 5-hydroxytryptamine and injected in pregnant mice and neonatal rats for vital neutral red staining of lung slicesas a standard in citrate-acetate buffer for its use in high performance liquid chromatography (HPLC) analysis

General Description

Colorless to pale pink crystals.

Air & Water Reactions

L-5-Hydroxytryptophan is sensitive to air. Slightly water soluble .

Reactivity Profile

L-5-Hydroxytryptophan reacts with bases. .

Fire Hazard

Flash point data for L-5-Hydroxytryptophan are not available, however, L-5-Hydroxytryptophan is probably combustible.

Biochem/physiol Actions

Hydroxylation of L-tryptophan occurs in the brain during serotonin synthesis. This is a step that controls the production of serotonin and also yields 5-hydroxy-l-tryptophan (5-HTP) as a metabolite. Thus, consumption 5-HTP, increases the serotonin level, which might result in depression, lack of sleep and severe headache.

Purification Methods

Likely impurities are 5-hydroxy-D-tryptophan and 5-benzyloxytryptophan. Crystallise 5-hydroxy-L-tryptophan under nitrogen from water by adding EtOH. Store it under nitrogen. Also dissolve it in the minimum volume of hot H2O (~0.7g in 4mL) under nitrogen (charcoal) and allowed it to crystallise at 5o. The picrolonate crystallises from H2O with m 184-186o(dec). [Greenstein & Winitz The Chemistry of the Amino Acids J. Wiley, Vol 3 p 2732-2737 1961, Morris & Armstrong J Org Chem 22 306 1957, Beilstein 22/14 V 278.]

Check Digit Verification of cas no

The CAS Registry Mumber 4350-09-8 includes 7 digits separated into 3 groups by hyphens. The first part of the number,starting from the left, has 4 digits, 4,3,5 and 0 respectively; the second part has 2 digits, 0 and 9 respectively.
Calculate Digit Verification of CAS Registry Number 4350-09:
(6*4)+(5*3)+(4*5)+(3*0)+(2*0)+(1*9)=68
68 % 10 = 8
So 4350-09-8 is a valid CAS Registry Number.
InChI:InChI=1/C11H12N2O3/c12-9(11(15)16)3-6-5-13-10-2-1-7(14)4-8(6)10/h1-2,4-5,9,13-14H,3,12H2,(H,15,16)

4350-09-8 Well-known Company Product Price

  • Brand
  • (Code)Product description
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  • Detail
  • TCI America

  • (H0531)  5-Hydroxy-L-tryptophan  >98.0%(HPLC)(T)

  • 4350-09-8

  • 1g

  • 230.00CNY

  • Detail
  • TCI America

  • (H0531)  5-Hydroxy-L-tryptophan  >98.0%(HPLC)(T)

  • 4350-09-8

  • 5g

  • 740.00CNY

  • Detail
  • Alfa Aesar

  • (A13954)  L-5-Hydroxytryptophan hydrate, 98%   

  • 4350-09-8

  • 0.25g

  • 177.0CNY

  • Detail
  • Alfa Aesar

  • (A13954)  L-5-Hydroxytryptophan hydrate, 98%   

  • 4350-09-8

  • 1g

  • 328.0CNY

  • Detail
  • Alfa Aesar

  • (A13954)  L-5-Hydroxytryptophan hydrate, 98%   

  • 4350-09-8

  • 5g

  • 1390.0CNY

  • Detail
  • Sigma

  • (H9772)  5-Hydroxy-L-tryptophan  powder

  • 4350-09-8

  • H9772-100MG

  • 402.48CNY

  • Detail
  • Sigma

  • (H9772)  5-Hydroxy-L-tryptophan  powder

  • 4350-09-8

  • H9772-250MG

  • 617.76CNY

  • Detail
  • Sigma

  • (H9772)  5-Hydroxy-L-tryptophan  powder

  • 4350-09-8

  • H9772-1G

  • 1,048.32CNY

  • Detail
  • Sigma

  • (H9772)  5-Hydroxy-L-tryptophan  powder

  • 4350-09-8

  • H9772-5G

  • 4,214.34CNY

  • Detail
  • Sigma

  • (H9772)  5-Hydroxy-L-tryptophan  powder

  • 4350-09-8

  • H9772-25G

  • 7,318.35CNY

  • Detail
  • Aldrich

  • (107751)  5-Hydroxy-L-tryptophan  98% (calc. on dried substance)

  • 4350-09-8

  • 107751-1G

  • 260.91CNY

  • Detail
  • USP

  • (1329913)  5-Hydroxy-L-tryptophan  United States Pharmacopeia (USP) Reference Standard

  • 4350-09-8

  • 1329913-100MG

  • 4,647.24CNY

  • Detail

4350-09-8SDS

SAFETY DATA SHEETS

According to Globally Harmonized System of Classification and Labelling of Chemicals (GHS) - Sixth revised edition

Version: 1.0

Creation Date: Aug 11, 2017

Revision Date: Aug 11, 2017

1.Identification

1.1 GHS Product identifier

Product name 5-hydroxy-L-tryptophan

1.2 Other means of identification

Product number -
Other names L-Tryptophan, 5-hydroxy-

1.3 Recommended use of the chemical and restrictions on use

Identified uses For industry use only.
Uses advised against no data available

1.4 Supplier's details

1.5 Emergency phone number

Emergency phone number -
Service hours Monday to Friday, 9am-5pm (Standard time zone: UTC/GMT +8 hours).

More Details:4350-09-8 SDS

4350-09-8Synthetic route

indol-5-ol
1953-54-4

indol-5-ol

L-serin
56-45-1

L-serin

L-5-HTP
4350-09-8

L-5-HTP

Conditions
ConditionsYield
Stage #1: indol-5-ol; L-serin With pyridoxal 5'-phosphate; thermotoga maritima with tryptophan synthase β-subunit from pyrococcus furiosus M145T N167D mutant In aq. phosphate buffer; water; dimethyl sulfoxide at 75℃; for 2h; pH=8; Sealed tube; Enzymatic reaction;
Stage #2: With hydrogenchloride In water
93%
With transformed Escherichia coli cells at 37℃; for 24h;70 % Chromat.
With tryptophan synthase; pyridoxal 5'-phosphate at 37℃; for 24h; pH=7.8; Enzymatic reaction;
DL-5-hydroxytryptophane amide
90830-06-1

DL-5-hydroxytryptophane amide

L-5-HTP
4350-09-8

L-5-HTP

Conditions
ConditionsYield
43%
43%
L-Tryptophan
73-22-3

L-Tryptophan

A

7-Hydroxy-L-tryptophan
25198-02-1

7-Hydroxy-L-tryptophan

B

4-Hydroxy-L-tryptophan
25242-90-4

4-Hydroxy-L-tryptophan

C

(S)-6-hydroxytryptophan
13567-14-1

(S)-6-hydroxytryptophan

D

L-5-HTP
4350-09-8

L-5-HTP

Conditions
ConditionsYield
With dihydrogen peroxide; edetate disodium; iron(II) sulfate; ascorbic acid In water at 22℃; for 0.0416667h;A n/a
B 10%
C n/a
D 12%
L-N-benzyloxycarbonyl-5-benzyloxytryptophan
3520-59-0

L-N-benzyloxycarbonyl-5-benzyloxytryptophan

L-5-HTP
4350-09-8

L-5-HTP

Conditions
ConditionsYield
With ethanol; palladium Hydrogenation;
5-benzyloxy-Nα-benzyloxycarbonyl-DL-tryptophan
3017-27-4

5-benzyloxy-Nα-benzyloxycarbonyl-DL-tryptophan

L-5-HTP
4350-09-8

L-5-HTP

Conditions
ConditionsYield
Multi-step reaction with 2 steps
1: quinine
2: palladium; ethanol / Hydrogenation
View Scheme
L-Tryptophan
73-22-3

L-Tryptophan

A

tryptamine
61-54-1

tryptamine

B

L-kynurenine
2922-83-0

L-kynurenine

C

hydroxyhexahydropyrrolo[2,3-b]indole-2-carboxylic acid

hydroxyhexahydropyrrolo[2,3-b]indole-2-carboxylic acid

D

(S )-2-Amino-4-(2-formamidophenyl)-4-oxobutanoic acid

(S )-2-Amino-4-(2-formamidophenyl)-4-oxobutanoic acid

E

hydroxyoxoindolylalanine

hydroxyoxoindolylalanine

F

2-amino-3-(2-oxoindolin-3-yl)propanoic acid
236094-74-9

2-amino-3-(2-oxoindolin-3-yl)propanoic acid

G

L-5-HTP
4350-09-8

L-5-HTP

Conditions
ConditionsYield
With dihydrogen peroxide at 37℃; for 6h; pH=4.6; Darkness; aq. borate buffer;
L-Tryptophan
73-22-3

L-Tryptophan

L-5-HTP
4350-09-8

L-5-HTP

Conditions
ConditionsYield
With tryptophan hydroxylase Enzymatic reaction;
With ferrous ammonium sulphate; 6-methyl-5,6,7,8-tetrahydrobopterin; tryptophan hydroxylase; oxygen In aq. buffer at 20℃; for 0.333333h; pH=7.7; Kinetics; Mechanism; Catalytic behavior; Reagent/catalyst; Solvent; Temperature; Enzymatic reaction; chemoselective reaction;
methanol
67-56-1

methanol

L-5-HTP
4350-09-8

L-5-HTP

5-hydroxy-DL-tryptophan methyl ester hydrochloride

5-hydroxy-DL-tryptophan methyl ester hydrochloride

Conditions
ConditionsYield
With thionyl chloride at -5 - 20℃; for 16h;98%
With thionyl chloride at 0 - 20℃; Inert atmosphere;74%
L-5-HTP
4350-09-8

L-5-HTP

3-(2-aminoethyl)-1H-indol-5-ol
50-67-9

3-(2-aminoethyl)-1H-indol-5-ol

Conditions
ConditionsYield
With pyridoxal 5'-phosphate; aromatic L-amino acid decarboxylase In various solvent(s) at 30℃; for 48h;97%
With NH4OH-NH4Cl buffer; pyridoxal 5'-phosphate at 30℃; for 0.5h; relative rate of CO2 evolution by aromatic L-amino acid decarboxylase from Micrococcus percitreus;
With isopropyl β-D-thiogalactoside; L-tryptophan decarboxylase in recombinant Escherichia coli at 28℃;
9-methoxy-9-BBN
38050-71-4

9-methoxy-9-BBN

L-5-HTP
4350-09-8

L-5-HTP

C19H25BN2O3

C19H25BN2O3

Conditions
ConditionsYield
With pyridine97%
methanol
67-56-1

methanol

L-5-HTP
4350-09-8

L-5-HTP

2-amino-3-(5-hydroxy-1H-indol-3-yl)propionic acid methyl ester
79812-02-5

2-amino-3-(5-hydroxy-1H-indol-3-yl)propionic acid methyl ester

Conditions
ConditionsYield
With thionyl chloride at 0 - 40℃; for 10h;95%
With thionyl chloride at -5 - 35℃; for 5h;92.9%
With hydrogenchloride at 0 - 20℃; for 18h; Fischer esterification;
copper(ll) bromide
7789-45-9

copper(ll) bromide

L-5-HTP
4350-09-8

L-5-HTP

[copper(II)(L-5-hydroxytryptophan)2(bromide)2] monohydrate

[copper(II)(L-5-hydroxytryptophan)2(bromide)2] monohydrate

Conditions
ConditionsYield
In methanol for 3h; Reflux;89%
3,4-dichlorophenylisocyanate
102-36-3

3,4-dichlorophenylisocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(3,4-dichlorophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(3,4-dichlorophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 2.5h;89%
di-tert-butyl dicarbonate
24424-99-5

di-tert-butyl dicarbonate

L-5-HTP
4350-09-8

L-5-HTP

(S)-2-tert-butoxycarbonylamino-3-(5-tert-butoxycarbonyloxy-1H-indol-3-yl)propionic acid
390816-59-8

(S)-2-tert-butoxycarbonylamino-3-(5-tert-butoxycarbonyloxy-1H-indol-3-yl)propionic acid

Conditions
ConditionsYield
With potassium hydroxide In isopropyl alcohol for 5h; pH=11.5 - 12.0;87%
nickel(II) bromide trihydrate

nickel(II) bromide trihydrate

L-5-HTP
4350-09-8

L-5-HTP

[nickel(II)(L-5-hydroxytryptophan)2(methanol)2(bromide)2]

[nickel(II)(L-5-hydroxytryptophan)2(methanol)2(bromide)2]

Conditions
ConditionsYield
In methanol for 3h; Reflux;86%
2,6-difluorophenylisothiocyanate
207974-17-2

2,6-difluorophenylisothiocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(2,6-difluorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(2,6-difluorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 3h;85%
2,4-diflurophenylisocyanate
59025-55-7

2,4-diflurophenylisocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(2,4-difluorophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(2,4-difluorophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 4h;85%
copper(II) choride dihydrate

copper(II) choride dihydrate

L-5-HTP
4350-09-8

L-5-HTP

[copper(II)(L-5-hydroxytryptophan)2(chloride)2]

[copper(II)(L-5-hydroxytryptophan)2(chloride)2]

Conditions
ConditionsYield
In methanol for 3h; Reflux;84.8%
nickel(II) nitrate hexahydrate

nickel(II) nitrate hexahydrate

L-5-HTP
4350-09-8

L-5-HTP

[nickel(II)(L-5-hydroxytryptophan)2(nitrate)2] methanol disolvate monohydrate

[nickel(II)(L-5-hydroxytryptophan)2(nitrate)2] methanol disolvate monohydrate

Conditions
ConditionsYield
In methanol for 3h; Reflux;84%
3,4-dichlorophenyl isothiocyanate
6590-94-9

3,4-dichlorophenyl isothiocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(3,4-dichlorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(3,4-dichlorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 4h;83%
cobalt(II) chloride hexahydrate

cobalt(II) chloride hexahydrate

L-5-HTP
4350-09-8

L-5-HTP

[cobalt(II)(L-5-hydroxytryptophan)2(chloride)2] pentahydrate

[cobalt(II)(L-5-hydroxytryptophan)2(chloride)2] pentahydrate

Conditions
ConditionsYield
In methanol for 3h; Reflux;82.8%
cobalt(II) bromide

cobalt(II) bromide

L-5-HTP
4350-09-8

L-5-HTP

[cobalt(II)(L-5-hydroxytryptophan)2(bromide)2]

[cobalt(II)(L-5-hydroxytryptophan)2(bromide)2]

Conditions
ConditionsYield
In methanol for 3h; Reflux;82.6%
nickel(II) chloride hexahydrate

nickel(II) chloride hexahydrate

L-5-HTP
4350-09-8

L-5-HTP

[nickel(II)(L-5-hydroxytryptophan)2(methanol)2(chloride)2] monohydrate

[nickel(II)(L-5-hydroxytryptophan)2(methanol)2(chloride)2] monohydrate

Conditions
ConditionsYield
In methanol for 3h; Reflux;82.6%
copper(II) nitrate trihydrate

copper(II) nitrate trihydrate

L-5-HTP
4350-09-8

L-5-HTP

[copper(II)(L-5-hydroxytryptophan)2(nitrate)2] monohydrate

[copper(II)(L-5-hydroxytryptophan)2(nitrate)2] monohydrate

Conditions
ConditionsYield
In methanol for 3h; Reflux;82.6%
methanol
67-56-1

methanol

di-tert-butyl dicarbonate
24424-99-5

di-tert-butyl dicarbonate

L-5-HTP
4350-09-8

L-5-HTP

(S)-methyl 2-((tert-butoxycarbonyl)amino)-3-(5-hydroxy-1H-indol-3-yl)propanoate
203736-17-8

(S)-methyl 2-((tert-butoxycarbonyl)amino)-3-(5-hydroxy-1H-indol-3-yl)propanoate

Conditions
ConditionsYield
Stage #1: methanol; L-5-HTP With thionyl chloride at 0 - 20℃; for 12h;
Stage #2: di-tert-butyl dicarbonate With triethylamine In dichloromethane at 0 - 20℃; for 6h;
82%
4-bromophenyl isocyanate
2493-02-9

4-bromophenyl isocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(4-bromophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(4-bromophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 3h;82%
3-indolylglyoxal
7269-72-9

3-indolylglyoxal

L-5-HTP
4350-09-8

L-5-HTP

6-hydroxy-1-(1H-indole-3-carbonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid
1350653-01-8

6-hydroxy-1-(1H-indole-3-carbonyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid

Conditions
ConditionsYield
Stage #1: 3-indolylglyoxal; L-5-HTP With sulfuric acid In water at 20℃; for 10h; Pictet-Spengler cyclisation;
Stage #2: With ammonia In water at 0℃; for 12h; pH=6 - 7;
80%
1-isothiocyanato-3-trifluoromethyl-benzene
1840-19-3

1-isothiocyanato-3-trifluoromethyl-benzene

L-5-HTP
4350-09-8

L-5-HTP

3-(5-hydroxy-1H-indol-3-yl)-2-(3-(3-trifluoromethylphenyl)thioureido)propanoic acid

3-(5-hydroxy-1H-indol-3-yl)-2-(3-(3-trifluoromethylphenyl)thioureido)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 3h;80%
4-fluorophenyl isothiocyanate
1544-68-9

4-fluorophenyl isothiocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(4-fluorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(4-fluorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran79%
3-chloro-4-fluorophenyl isothiocyanate
137724-66-4

3-chloro-4-fluorophenyl isothiocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(3-chloro-4-fluorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(3-chloro-4-fluorophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 4h;79%
3-bromophenyl isothiocyanate
2131-59-1

3-bromophenyl isothiocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(3-bromophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(3-bromophenyl)thioureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 3.5h;78%
zinc dibromide

zinc dibromide

L-5-HTP
4350-09-8

L-5-HTP

[zinc(II)(L-5-hydroxytryptophan)2(bromide)2] monohydrate

[zinc(II)(L-5-hydroxytryptophan)2(bromide)2] monohydrate

Conditions
ConditionsYield
In methanol for 3h; Reflux;77.2%
1,3-dimethylbarbituric acid
769-42-6

1,3-dimethylbarbituric acid

L-5-HTP
4350-09-8

L-5-HTP

5-hydroxy-L-tryptophan 1,3-dimethylbarbituric acid complex

5-hydroxy-L-tryptophan 1,3-dimethylbarbituric acid complex

Conditions
ConditionsYield
In water at 100℃; for 0.5h;77%
di-tert-butyl dicarbonate
24424-99-5

di-tert-butyl dicarbonate

L-5-HTP
4350-09-8

L-5-HTP

(S)-2-((tert-butoxycarbonyl)amino)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid
119768-45-5

(S)-2-((tert-butoxycarbonyl)amino)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With potassium carbonate In tetrahydrofuran; water at 20℃; for 2h;76%
Stage #1: di-tert-butyl dicarbonate; L-5-HTP With sodium hydrogencarbonate In water at 0 - 35℃; for 24h;
Stage #2: With citric acid In water pH=6;
58.16%
With triethylamine In 1,4-dioxane at 20℃;
[Ru(η(6)-cymene)(acetone)3](CF3SO3)2

[Ru(η(6)-cymene)(acetone)3](CF3SO3)2

trifluoroacetic acid
76-05-1

trifluoroacetic acid

L-5-HTP
4350-09-8

L-5-HTP

[(η6-p-cymene)Ru(η6-5-hydroxytryptophan)](CF3SO3)2*CF3COOH

[(η6-p-cymene)Ru(η6-5-hydroxytryptophan)](CF3SO3)2*CF3COOH

Conditions
ConditionsYield
In trifluoroacetic acid ligand was added to Ru-complex in CF3COOH, stirred for 12 h at 50°C; pptd. with Et2O, dried in vac.; elem. anal.;75%
p-chlorphenylisocyanate
104-12-1

p-chlorphenylisocyanate

L-5-HTP
4350-09-8

L-5-HTP

2-(3-(4-chlorophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

2-(3-(4-chlorophenyl)ureido)-3-(5-hydroxy-1H-indol-3-yl)propanoic acid

Conditions
ConditionsYield
With pyridine; triethylamine In tetrahydrofuran for 3.5h;75%

4350-09-8Relevant articles and documents

Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer

Ohler, Amanda,Long, Hanna,Ohgo, Kei,Tyson, Kristin,Murray, David,Davis, Amanda,Whittington, Chris,Hvastkovs, Eli G.,Duffy, Liam,Haddy, Alice,Sargent, Andrew L.,Allen, William E.,Offenbacher, Adam R.

supporting information, p. 3107 - 3110 (2021/04/02)

Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into a β-hairpin peptide and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function of 5HOW as probes for biological electron transfer.

Mutagenesis of an Active-Site Loop in Tryptophan Hydroxylase Dramatically Slows the Formation of an Early Intermediate in Catalysis

Subedi, Bishnu P.,Fitzpatrick, Paul F.

supporting information, p. 5185 - 5192 (2018/04/23)

Solution studies of the aromatic amino acid hydroxylases are consistent with the FeIVO intermediate not forming until both the amino acid and tetrahydropterin substrates have bound. Structural studies have shown that the positions of active-site loops differs significantly between the free enzyme and the enzyme-amino acid-tetrahydropterin complex. In tryptophan hydroxylase (TrpH) these mobile loops contain residues 124-134 and 365-371, with a key interaction involving Ile366. The I366N mutation in TrpH results in decreases of 1-2 orders of magnitude in the kcat and kcat/Km values. Single turnover analyses establish that the limiting rate constant for turnover is product release for the wild-type enzyme but is formation of the first detectable intermediate I in catalysis in the mutant enzyme. The mutation does not alter the kinetics of NO binding to the ternary complex nor does it uncouple FeIVO formation from amino acid hydroxylation. The effects on the kcat value of wild-type TrpH of changing viscosity are consistent with rate-limiting product release. While the effect of viscosity on the kcat/KO2 value is small, consistent with reversible oxygen binding, the effects on the kcat/Km values for tryptophan and the tetrahydropterin are large, with the latter value exceeding the expected limit and varying with the identity of the viscogen. In contrast, the kinetic parameters of I366N TrpH show small changes with viscosity. The results are consistent with binding of the amino acid and pterin substrate to form the ternary complex being directly coupled to closure of loops over the active site and formation of the reactive complex. The mutation destabilizes this initial event.

A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation

Murciano-Calles, Javier,Romney, David K.,Brinkmann-Chen, Sabine,Buller, Andrew R.,Arnold, Frances H.

supporting information, p. 11577 - 11581 (2016/10/24)

Naturally occurring enzyme homologues often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homologue. A small set of mutations of the tryptophan synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimics the activation afforded by binding of the α-subunit, was demonstrated to have a similar activating effect in different TrpB homologues with as little as 57 % sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism: mimicry of α-subunit binding. From these enzymes, we identified a new TrpB catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans. This demonstrates that allosteric activation can be recapitulated throughout a protein family to explore natural sequence diversity for desirable biocatalytic transformations.

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