Literature summary for 2.1.1.28 extracted from

Gee, C.L.; Nourse, A.; Hsin, A.Y.; Wu, Q.; Tyndall, J.D.; Grunewald, G.L.; McLeish, M.J.; Martin, J.L.
Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active (2005), Biochim. Biophys. Acta, 1750, 82-92.

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli strain BL21(DE3)pLysS	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
hanging drop vapour diffusion method is used with 2 mircol drops on 3 M sealed over 500 mircol or 100 microl precipitant in 24-well or 96-well trays. In the absence of reducing agents crystals grow on protein concentrations of 30 to 40 mg/ml and appear in two or three days. The addition of DTT inhibits formation of crystals under the same condition. Reduced and oxidized glutathione are added to PEG/LiCl crystallisation conditions, crystals only grow in drops where the amount of oxidized glutathione is higher than reduced, the ratio of 1:20 (reduced glutathione/oxidized glutathione) gives the largest crystals.	Homo sapiens

Crystallization (Comment)

Organism

hanging drop vapour diffusion method is used with 2 mircol drops on 3 M sealed over 500 mircol or 100 microl precipitant in 24-well or 96-well trays. In the absence of reducing agents crystals grow on protein concentrations of 30 to 40 mg/ml and appear in two or three days. The addition of DTT inhibits formation of crystals under the same condition. Reduced and oxidized glutathione are added to PEG/LiCl crystallisation conditions, crystals only grow in drops where the amount of oxidized glutathione is higher than reduced, the ratio of 1:20 (reduced glutathione/oxidized glutathione) gives the largest crystals.

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
C139A	the C139A mutant separates into two forms on gel filtration, consistent with a monomer and dimer form, the equilibrium is significantly shifted in favour of the monomer. C139A mutant has similar kinetic constants as the wild type human phenylethanolamine N-methyltransferase	Homo sapiens
C48A	the C48A mutant separates into two forms on gel filtration, consistent with a monomer and dimer form, the equilibrium is significantly shifted in favour of the monomer. C48A mutant has similar kinetic constants as the wild type human phenylethanolamine N-methyltransferase. The monomer-dimer equilibrium in analytical ultracentrifugation for the dimer fractions of phenylethanolamine N-methyltransferase-His and C48A phenylethanolamine N-methyltransferase is 10fold lower than for the monomer fractions (Kd 35-64 microM and 305-551 microM, respectively). The relative Kd values show that C48A phenylethanolamine N-methyltransferase is less likely to form dimer than phenylethanolamine N-methyltransferase-His.	Homo sapiens
C48A/C139A	similar kinetic constants as the wild type human phenylethanolamine N-methyltransferase	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.0035	-	S-adenosyl-L-methionine	purified in the absence of reducing agent, dimer, His-tagged	Homo sapiens
0.095	-	phenylethanolamine	purified in the absence of reducing agent, dimer, His-tagged	Homo sapiens

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
30000	-	SDS-PAGE	Homo sapiens
30280	-	analytical ultracentrifugation, non tagged phenylethanolamine N-methyltransferase, monomer	Homo sapiens
60550	-	analytical ultracentrifugation, non tagged phenylethanolamine N-methyltransferase, dimer	Homo sapiens

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
6X-histidine tagged phenylethanolamine N-methyltransferase purified on a 10 ml column and with gel filtration. Non-tagged phenylethanolamine N-methyltransferase is purified with DEAE-Sepharose and gel filtration.	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
S-adenosyl-L-methionine + phenylethanolamine	-	Homo sapiens	S-adenosyl-L-homocysteine + N-methylphenylethanolamine	-	?

Subunits

Subunits	Comment	Organism
dimer	gel filtration reveals the presence of two species at elution volumes consistent with monomeric and dimeric human phenylethanolamine N-methyltransferase. The more prominent peak corresponds to the dimer form. The amount of dimer can be reduced either by using a more elute concentration of the protein or by the addition of 0.5 mM DTT to the running buffer. SDS-PAGE can not distinguish between the two forms. Native PAGE clearly distinguishes between the two forms of human phenylethanolamine N-methyltransferase. Crystals from the dimer fraction grow faster. Monomer and dimer human phenylethanolamine N-methyltransferase have similar kinetic constatns. The monomer-dimer equilibruim in analytical ultracentrifugation for the dimer frations of phenylethanolamine N-methyltransferase-His and C48A phenylethanolamine N-methyltransferase is 10fold lower than for the monomer fractions (Kd 35-64 microM and 305-551 microM, respectively).	Homo sapiens

Synonyms

Synonyms	Comment	Organism
hPNMT	-	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.045	-	phenylethanolamine	purified in the absence of reducing agent, dimer, His-tagged	Homo sapiens